Polynucleotide synthesis method, system and kit

ABSTRACT

The invention relates to new methods for synthesising polynucleotide molecules according to a predefined nucleotide sequence. The invention also relates to methods for the assembly of synthetic polynucleotides following synthesis, as well as systems and kits for performing the synthesis and/or assembly methods.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational application number PCT/GB2019/051423, filed May 23, 2019,which claims the benefit of Great Britain application number GB1808474.9, filed May 23, 2018, the entire contents of each of which isincorporated by reference.

FIELD OF THE INVENTION

The invention relates to new methods for synthesising polynucleotidemolecules according to a predefined nucleotide sequence. The inventionalso relates to methods for the assembly of synthetic polynucleotidesfollowing synthesis, as well as systems and kits for performing thesynthesis and/or assembly methods.

BACKGROUND TO THE INVENTION

Two primary methods exist for the synthesis and assembly ofpolynucleotide molecules, particularly DNA.

Phosphoramidite chemistry is a synthetic approach that assemblesmonomers of chemically activated T, C, A or G into oligonucleotides ofapproximately 100/150 bases in length via a stepwise process. Thechemical reaction steps are highly sensitive and the conditionsalternate between fully anhydrous (complete absence of water), aqueousoxidative and acidic (Roy and Caruthers, Molecules, 2013, 18,14268-14284). If the reagents from the previous reaction step have notbeen completely removed this will be detrimental to future steps ofsynthesis. Accordingly, this synthesis method is limited to theproduction of polynucleotides of length of approximately 100nucleotides.

The Polymerase Synthetic approach uses a polymerase to synthesise acomplementary strand to a DNA template using T, C, A and Gtriphosphates. The reaction conditions are aqueous and mild and thisapproach can be used to synthesise DNA polynucleotides which are manythousands of bases in length. The main disadvantage of this method isthat single- and double-stranded DNA cannot be synthesised de novo bythis method, it requires a DNA template from which a copy is made.(Kosuri and Church, Nature Methods, 2014, 11, 499-507).

Thus previous methods cannot be used to synthesise double-stranded DNAde novo without the aid of a pre-existing template molecule which iscopied.

The inventors have developed new methodologies by which single- anddouble-stranded polynucleotide molecules can be synthesised de novo in astepwise manner without the need to copy a pre-existing templatemolecule. Such methods also avoid the extreme conditions associated withphosphoramidite chemistry techniques and in contrast are carried outunder mild, aqueous conditions around neutral pH. Such methods alsoenable de novo synthesis of single- or double-stranded polynucleotidemolecules with a potential 10⁸ improvement on current synthesis methodswith nucleotide lengths of >100mers to full genomes, providing a widerange of possibly applications in synthetic biology.

SUMMARY OF THE INVENTION

The invention provides an in vitro method of synthesising adouble-stranded polynucleotide having a predefined sequence, the methodcomprising performing cycles of synthesis wherein each cycle comprisescleaving a double-stranded polynucleotide and extending the cleaveddouble-stranded polynucleotide by incorporating a nucleotide pair,wherein a terminal end of a first strand of the cleaved double-strandedpolynucleotide is extended by the addition of a nucleotide of thepredefined sequence and a terminal end of the second strand of thecleaved double-stranded polynucleotide which is hybridized to the firststrand is extended by the addition of a partner nucleotide therebyforming a nucleotide pair with the incorporated nucleotide of the firststrand. Preferably, the methods are for synthesising DNA.

In any of the methods of the invention described herein the methods mayprovide for the synthesis of a single-stranded polynucleotide moleculewherein following synthesis of the double-stranded polynucleotidemolecule having a predefined sequence one strand of the double-strandedpolynucleotide molecule is removed, or copied and/or amplified, toprovide the single-stranded polynucleotide molecule.

In any of the methods of the invention described herein the methods mayprovide for the synthesis of a double-stranded or single-strandedoligonucleotide. Thus all references herein to the synthesis of adouble-stranded or single-stranded polynucleotide using any of themethods of the invention apply mutatis mutandis to the synthesis of adouble-stranded or single-stranded oligonucleotide.

In methods of the invention described above, each cycle may compriseextending the first strand by adding the nucleotide of the predefinedsequence together with an attached reversible blocking group followed byextending the second strand, wherein the reversible blocking group isremoved before or after the second strand is extended. In any suchmethod, in each cycle the nucleotides may be incorporated into a cleavedscaffold polynucleotide.

The invention provides a method as described above and herein, whereineach cycle comprises:

-   -   (1) providing a scaffold polynucleotide;    -   (2) cleaving the scaffold polynucleotide at a cleavage site;    -   (3) adding to the cleaved scaffold polynucleotide by the action        of a nucleotide transferase or polymerase enzyme a nucleotide of        the predefined sequence, the nucleotide comprising a reversible        terminator group which prevents further extension by the enzyme;    -   (4) removing the reversible terminator group from the nucleotide        of the predefined sequence; and    -   (5) ligating a ligation polynucleotide to the cleaved scaffold        polynucleotide, the ligation polynucleotide comprising a partner        nucleotide for the nucleotide of the predefined sequence,        wherein upon ligation the nucleotide of the predefined sequence        pairs with the partner nucleotide.

In methods involving a scaffold polynucleotide the scaffoldpolynucleotide may be provided comprising a synthesis strand and asupport strand hybridized thereto, wherein the synthesis strandcomprises a primer strand portion and a helper strand portion. In anysuch methods the helper strand portion may be removed from the scaffoldpolynucleotide prior to any one, more or all cleavage steps.

In any such method involving a scaffold polynucleotide the synthesisstrand may be the first strand and the support strand may be the secondstrand.

In any such method the support strand may be extended by ligating to thesupport strand a ligation polynucleotide, wherein in each cycle ofsynthesis the ligation polynucleotide comprises the nucleotide formingthe nucleotide pair with the predefined nucleotide incorporated into thefirst strand in that cycle.

The ligation polynucleotide may be single-stranded or double-stranded.Preferably, the ligation polynucleotide is double-stranded.

In methods wherein the ligation polynucleotide is double-stranded, theligation polynucleotide may preferably comprise a support strand and ahelper strand. The helper strand may be removed from the scaffoldpolynucleotide before the step of cleavage in the next synthesis cycle,in such methods the helper strand is removed after the ligation step.

The invention provides a method as described above, wherein step (1)comprises providing a scaffold polynucleotide comprising a synthesisstrand and a support strand hybridized thereto, wherein the synthesisstrand comprises a primer strand portion, and the support strandcomprises a universal nucleotide; wherein step (2) comprises cleavingthe scaffold polynucleotide at a cleavage site, the site defined by asequence comprising the universal nucleotide in the support strand,wherein cleavage comprises cleaving the support strand and removing theuniversal nucleotide from the scaffold polynucleotide; and wherein instep (5) the ligation polynucleotide comprises a support strandcomprising the partner nucleotide and a universal nucleotide whichdefines a cleavage site for use in the next cycle, and wherein theligation polynucleotide is ligated to the support strand of the cleavedscaffold polynucleotide thereby forming the nucleotide pair.

The invention provides a method as described above, comprising:

-   -   (1) providing a scaffold polynucleotide comprising a synthesis        strand and a support strand hybridized thereto, wherein the        synthesis strand comprises a primer strand portion and a helper        strand portion separated by a single-strand break, and the        support strand comprises a universal nucleotide;    -   (2) cleaving the scaffold polynucleotide at a cleavage site, the        site defined by a sequence comprising the universal nucleotide        in the support strand, wherein cleavage comprises cleaving the        support strand and removing the universal nucleotide from the        scaffold polynucleotide to provide a cleaved double-stranded        scaffold polynucleotide comprising a support strand and a        synthesis strand comprising the primer strand portion;    -   (3) extending the terminal end of the primer strand portion of        the synthesis strand of the cleaved double-stranded scaffold        polynucleotide with a first nucleotide of the predefined        sequence by the action of a nucleotide transferase or polymerase        enzyme, the first nucleotide comprising a reversible terminator        group which prevents further extension by the enzyme;    -   (4) removing the terminator group from the first nucleotide;    -   (5) ligating a double-stranded ligation polynucleotide to the        cleaved scaffold polynucleotide, the ligation polynucleotide        comprising a support strand and a helper strand hybridised        thereto and further comprising a complementary ligation end, the        ligation end comprising:        -   (i) in the support strand a universal nucleotide and a            partner nucleotide for the first nucleotide, wherein the            partner nucleotide for the first nucleotide overhangs the            helper strand; and        -   (ii) in the helper strand a terminal nucleotide lacking a            phosphate group;    -    wherein upon ligation of the support strands the first        nucleotide pairs with the partner nucleotide;    -   (6) cleaving the scaffold polynucleotide at a cleavage site, the        site defined by a sequence comprising the universal nucleotide        in the support strand, wherein cleavage comprises cleaving the        support strand and removing the universal nucleotide from the        scaffold polynucleotide to provide a cleaved double-stranded        scaffold polynucleotide comprising a support strand and a        synthesis strand comprising a primer strand portion;    -   (7) extending the terminal end of the primer strand portion of        the synthesis strand of the cleaved double-stranded scaffold        polynucleotide with the next nucleotide of the predefined        nucleotide sequence by the action of a nucleotide transferase or        polymerase enzyme, the next nucleotide comprising a reversible        terminator group which prevents further extension by the enzyme;    -   (8) removing the terminator group from the next nucleotide; and    -   (9) ligating a double-stranded ligation polynucleotide to the        cleaved scaffold polynucleotide, the ligation polynucleotide        comprising a support strand and a helper strand hybridised        thereto and further comprising a complementary ligation end, the        ligation end comprising:        -   (i) in the support strand a universal nucleotide and a            partner nucleotide for the next nucleotide, wherein the            partner nucleotide for the next nucleotide overhangs the            helper strand; and        -   (ii) in the helper strand a terminal nucleotide lacking a            phosphate group;    -    wherein upon ligation of the support strands the next        nucleotide pairs with the partner nucleotide;    -   (10) repeating steps 6 to 9 multiple times to provide the        double-stranded polynucleotide having a predefined nucleotide        sequence.

Any such method as described above may be performed according to amethod of the invention designated synthesis method version 1 wherein:

-   -   a) prior to and at the cleavage step of the first cycle (step 2)        the universal nucleotide occupies position n in the support        strand of the scaffold polynucleotide, wherein position n is the        nucleotide position in the support strand which is opposite the        position in the synthesis strand which will be occupied by the        first nucleotide of the predefined sequence upon its addition to        the terminal end of the primer strand portion in that cycle,        wherein the nucleotide at position n in the support strand is        opposite the terminal nucleotide of the helper strand and is        paired therewith;    -   b) in the cleavage step of the first cycle (step 2) the support        strand of the scaffold polynucleotide is cleaved between        positions n and n−1, wherein position n−1 is the next nucleotide        position in the support strand relative to position n in the        direction distal to the helper strand/proximal to the primer        strand portion;    -   c) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the partner nucleotide for the first        nucleotide of the predefined sequence is the terminal nucleotide        of the support strand and occupies position n, wherein the        universal nucleotide occupies position n+1 in the support strand        and is paired with the terminal nucleotide of the helper strand,        wherein position n is the nucleotide position which will be        opposite the first nucleotide of the predefined sequence upon        ligation of the ligation polynucleotide to the cleaved scaffold        polynucleotide in step 5;    -   d) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles:        -   i. the universal nucleotide occupies position n in the            support strand of the scaffold polynucleotide, wherein            position n is the nucleotide position in the support strand            which is opposite the position in the synthesis strand which            will be occupied by the next nucleotide of the predefined            sequence upon its addition to the terminal end of the primer            strand portion in that cycle; and        -   ii. the support strand of the scaffold polynucleotide is            cleaved between positions n and n−1, wherein n−1 is the next            nucleotide position in the support strand relative to            position n in the direction distal to the helper            strand/proximal to the primer strand portion; and    -   e) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the partner nucleotide for the next nucleotide of the        predefined sequence in that cycle is the terminal nucleotide of        the support strand and occupies position n, and the universal        nucleotide occupies position n+1 in the support strand and is        paired with the terminal nucleotide of the helper strand;        wherein position n is the nucleotide position which upon        ligation of the ligation polynucleotide to the cleaved scaffold        polynucleotide will be opposite the next nucleotide of the        predefined sequence incorporated in that cycle (step 7).

Alternatively, any such method as described above may be performedaccording to a method of the invention designated synthesis methodversion 2 wherein:

-   -   a) prior to and at the cleavage step of the first cycle (step 2)        the universal nucleotide occupies position n+1 in the support        strand of the scaffold polynucleotide, wherein position n is the        nucleotide position in the support strand which is opposite the        position in the synthesis strand which will be occupied by the        first nucleotide of the predefined sequence upon its addition to        the terminal end of the primer strand portion in that cycle,        wherein the nucleotide at position n in the support strand is        opposite the terminal nucleotide of the helper strand and is        paired therewith, and wherein n+1 is the next nucleotide        position in the support strand relative to position n in the        direction proximal to the helper strand/distal to the primer        strand portion;    -   b) in the cleavage step of the first cycle (step 2) the support        strand of the scaffold polynucleotide is cleaved between        positions n and n−1, wherein position n−1 is the next nucleotide        position in the support strand relative to position n in the        direction distal to the helper strand/proximal to the primer        strand portion;    -   c) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the partner nucleotide for the first        nucleotide of the predefined sequence is the terminal nucleotide        of the support strand and occupies position n, wherein the        universal nucleotide occupies position n+2 in the support strand        and is paired with the penultimate nucleotide of the helper        strand, wherein position n is the nucleotide position which will        be opposite the first nucleotide of the predefined sequence upon        ligation of the ligation polynucleotide to the cleaved scaffold        polynucleotide in step 5 and position n+2 is the second position        in the support strand relative to position n in the direction        proximal to the helper strand/distal to the primer strand        portion;    -   d) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles:        -   i. the universal nucleotide occupies position n+1 in the            support strand of the scaffold polynucleotide, wherein            position n is the nucleotide position in the support strand            which is opposite the position in the synthesis strand which            will be occupied by the next nucleotide of the predefined            sequence upon its addition to the terminal end of the primer            strand portion in that cycle, and wherein n+1 is the next            nucleotide position in the support strand relative to            position n in the direction proximal to the helper            strand/distal to the primer strand portion; and        -   ii. the support strand of the scaffold polynucleotide is            cleaved between positions n and n−1, wherein n−1 is the next            nucleotide position in the support strand relative to            position n in the direction distal to the helper            strand/proximal to the primer strand portion; and    -   e) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the partner nucleotide for the next nucleotide of the        predefined sequence in that cycle is the terminal nucleotide of        the support strand and occupies position n, and the universal        nucleotide occupies position n+2 in the support strand and is        paired with the penultimate nucleotide of the helper strand;        wherein position n is the nucleotide position which upon        ligation of the ligation polynucleotide to the cleaved scaffold        polynucleotide will be opposite the next nucleotide of the        predefined sequence incorporated in that cycle (step 7) and        position n+2 is the second position in the support strand        relative to position n in the direction proximal to the helper        strand/distal to the primer strand portion.

Alternatively still, any such method as described above may be performedaccording to a method of the invention designated synthesis methodversion 3 wherein:

-   -   a) prior to and at the cleavage step of the first cycle (step 2)        the universal nucleotide occupies position n in the support        strand of the scaffold polynucleotide, wherein position n is the        nucleotide position in the support strand which is opposite the        position in the synthesis strand which will be occupied by the        first nucleotide of the predefined sequence upon its addition to        the terminal end of the primer strand portion in that cycle,        wherein the nucleotide at position n in the support strand is        opposite the terminal nucleotide of the helper strand and is        paired therewith;    -   b) in the cleavage step of the first cycle (step 2) the support        strand of the scaffold polynucleotide is cleaved between        positions n−1 and n−2, wherein positions n−1 and n−2 are        respectively the next and subsequent nucleotide positions in the        support strand relative to position n in the direction distal to        the helper strand/proximal to the primer strand portion;    -   c) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the partner nucleotide for the first        nucleotide of the predefined sequence is the penultimate        nucleotide of the support strand and occupies position n,        wherein the universal nucleotide occupies position n+1 in the        support strand and is paired with the terminal nucleotide of the        helper strand, wherein position n is the nucleotide position        which will be opposite the first nucleotide of the predefined        sequence upon ligation of the ligation polynucleotide to the        cleaved scaffold polynucleotide in step 5, and wherein position        n+1 is the next nucleotide position in the support strand        relative to position n in the direction proximal to the helper        strand/distal to the primer strand portion;    -   d) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles:        -   i. the universal nucleotide occupies position n in the            support strand of the scaffold polynucleotide, wherein            position n is the nucleotide position in the support strand            which is opposite the position in the synthesis strand which            will be occupied by the next nucleotide of the predefined            sequence upon its addition to the terminal end of the primer            strand portion in that cycle; and        -   ii. the support strand of the scaffold polynucleotide is            cleaved between positions n−1 and n−2, wherein positions n−1            and n−2 are respectively the next and subsequent nucleotide            positions in the support strand relative to position n in            the direction distal to the helper strand/proximal to the            primer strand portion; and    -   e) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the partner nucleotide for the next nucleotide of the        predefined sequence in that cycle is the penultimate nucleotide        of the support strand and occupies position n, and the universal        nucleotide occupies position n+1 in the support strand and is        paired with the terminal nucleotide of the helper strand;        wherein position n is the nucleotide position which upon        ligation of the ligation polynucleotide to the cleaved scaffold        polynucleotide will be opposite the next nucleotide of the        predefined sequence incorporated in that cycle (step 7), and        wherein position n+1 is the next nucleotide position in the        support strand relative to position n in the direction proximal        to the helper strand/distal to the primer strand portion.

Alternatively still, any such method as described above may be performedaccording to a method of the invention designated synthesis methodversion 4 wherein:

-   -   a) prior to and at the cleavage step of the first cycle (step 2)        the universal nucleotide occupies position n+2 in the support        strand of the scaffold polynucleotide, wherein position n is the        nucleotide position in the support strand which is opposite the        position in the synthesis strand which will be occupied by the        first nucleotide of the predefined sequence upon its addition to        the terminal end of the primer strand portion in that cycle,        wherein the nucleotide at position n in the support strand is        opposite the terminal nucleotide of the helper strand and is        paired therewith, and wherein n+2 is the second nucleotide        position in the support strand relative to position n in the        direction proximal to the helper strand/distal to the primer        strand portion;    -   b) in the cleavage step of the first cycle (step 2) the support        strand of the scaffold polynucleotide is cleaved between        positions n and n−1, wherein position n−1 is the next nucleotide        position in the support strand relative to position n in the        direction distal to the helper strand/proximal to the primer        strand portion;    -   c) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the partner nucleotide for the first        nucleotide of the predefined sequence is the terminal nucleotide        of the support strand and occupies position n, wherein the        universal nucleotide occupies position n+3 in the support strand        and is paired with the nucleotide which is two positions removed        from the terminal nucleotide of the helper strand in the        direction distal to the primer strand portion, wherein position        n is the nucleotide position which will be opposite the first        nucleotide of the predefined sequence upon ligation of the        ligation polynucleotide to the cleaved scaffold polynucleotide        in step 5 and position n+3 is the third position in the support        strand relative to position n in the direction proximal to the        helper strand/distal to the primer strand portion;    -   d) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles:        -   i. the universal nucleotide occupies position n+2 in the            support strand of the scaffold polynucleotide, wherein            position n is the nucleotide position in the support strand            which is opposite the position in the synthesis strand which            will be occupied by the next nucleotide of the predefined            sequence upon its addition to the terminal end of the primer            strand portion in that cycle, and wherein n+2 is the second            nucleotide position in the support strand relative to            position n in the direction proximal to the helper            strand/distal to the primer strand portion; and        -   ii. the support strand of the scaffold polynucleotide is            cleaved between positions n and n−1, wherein position n−1 is            the next nucleotide position in the support strand relative            to position n in the direction distal to the helper            strand/proximal to the primer strand portion; and    -   e) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the partner nucleotide for the next nucleotide of the        predefined sequence in that cycle is the terminal nucleotide of        the support strand and occupies position n, and the universal        nucleotide occupies position n+3 in the support strand and is        paired with the nucleotide which is two positions removed from        the terminal nucleotide of the helper strand in the direction        distal to the primer strand portion; wherein position n is the        nucleotide position which upon ligation of the ligation        polynucleotide to the cleaved scaffold polynucleotide will be        opposite the next nucleotide of the predefined sequence        incorporated in that cycle (step 7) and position n+3 is the        third position in the support strand relative to position n in        the direction proximal to the helper strand/distal to the primer        strand portion.

Any such method may be performed according to a method of the inventionwhich is a variation of synthesis method version 4 wherein:

-   -   (i) in the cleavage step of the first cycle (step 2) the        universal nucleotide instead occupies position n+3 in the        support strand of the scaffold polynucleotide, wherein n+3 is        the third nucleotide position in the support strand relative to        position n in the direction proximal to the helper strand/distal        to the primer strand portion; and the support strand of the        scaffold polynucleotide is cleaved between positions n and n−1;    -   (ii) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the universal nucleotide instead occupies        position n+4 in the support strand and is paired with the        nucleotide which is 3 positions removed from the terminal        nucleotide of the helper strand at the complementary ligation        end; wherein position n+4 is position 4 in the support strand        relative to position n in the direction proximal to the helper        strand/distal to the primer strand portion;    -   (iii) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles the universal nucleotide        occupies position n+3 in the support strand of the scaffold        polynucleotide, and the support strand of the scaffold        polynucleotide is cleaved between positions n and n−1; and    -   (iv) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the universal nucleotide occupies position n+4 in the        support strand and is paired with the nucleotide which is 2        positions removed from the terminal nucleotide of the helper        strand at the complementary ligation end.

Any such method may be performed according to a method of the inventionwhich is a further variation of synthesis method version 4 wherein:

-   -   (i) in the cleavage step of the first cycle (step 2) the        universal nucleotide instead occupies position n+3+x in the        support strand of the scaffold polynucleotide, wherein n+3 is        the third nucleotide position in the support strand relative to        position n in the direction proximal to the helper strand/distal        to the primer strand portion; and the support strand of the        scaffold polynucleotide is cleaved between positions n and n−1;    -   (ii) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the universal nucleotide instead occupies        position n+4+x in the support strand and is paired with the        nucleotide which is 3+x positions removed from the terminal        nucleotide of the helper strand at the complementary ligation        end; wherein position n+4 is position 4 in the support strand        relative to position n in the direction proximal to the helper        strand/distal to the primer strand portion;    -   (iii) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles the universal nucleotide        occupies position n+3+x in the support strand of the scaffold        polynucleotide and the support strand of the scaffold        polynucleotide is cleaved between positions n and n−1;    -   (iv) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the universal nucleotide occupies position n+4+x in the        support strand and is paired with the nucleotide which is 2+x        positions removed from the terminal nucleotide of the helper        strand at the complementary ligation end; and    -   (v) wherein x is a whole number between 1 to 10 or more, and        wherein x is the same whole number in steps (2), (5), (6) and        (9).

Alternatively still, any such method as described above may be performedaccording to a method of the invention designated synthesis methodversion 5 wherein:

-   -   a) prior to and at the cleavage step of the first cycle (step 2)        the universal nucleotide occupies position n+1 in the support        strand of the scaffold polynucleotide, wherein position n is the        nucleotide position in the support strand which is opposite the        position in the synthesis strand which will be occupied by the        first nucleotide of the predefined sequence upon its addition to        the terminal end of the primer strand portion in that cycle,        wherein the nucleotide at position n in the support strand is        opposite the terminal nucleotide of the helper strand and is        paired therewith, and wherein n+1 is the next nucleotide        position in the support strand relative to position n in the        direction proximal to the helper strand/distal to the primer        strand portion;    -   b) in the cleavage step of the first cycle (step 2) the support        strand of the scaffold polynucleotide is cleaved between        positions n−1 and n−2, wherein positions n−1 and n−2 are        respectively the next and subsequent nucleotide positions in the        support strand relative to position n in the direction distal to        the helper strand/proximal to the primer strand portion;    -   c) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the partner nucleotide for the first        nucleotide of the predefined sequence is the penultimate        nucleotide of the support strand and occupies position n,        wherein the universal nucleotide occupies position n+2 in the        support strand and is paired with the penultimate nucleotide of        the helper strand, wherein position n is the nucleotide position        which will be opposite the first nucleotide of the predefined        sequence upon ligation of the ligation polynucleotide to the        cleaved scaffold polynucleotide, and wherein position n+2 is the        second nucleotide position in the support strand relative to        position n in the direction proximal to the helper strand/distal        to the primer strand portion;    -   d) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles:        -   i. the universal nucleotide occupies position n+1 in the            support strand of the scaffold polynucleotide, wherein            position n is the nucleotide position in the support strand            which is opposite the position in the synthesis strand which            will be occupied by the next nucleotide of the predefined            sequence upon its addition to the terminal end of the primer            strand portion in that cycle, and wherein n+1 is the next            nucleotide position in the support strand relative to            position n in the direction proximal to the helper            strand/distal to the primer strand portion; and        -   ii. the support strand of the scaffold polynucleotide is            cleaved between positions n−1 and n−2, wherein positions n−1            and n−2 are respectively the next and subsequent nucleotide            positions in the support strand relative to position n in            the direction distal to the helper strand/proximal to the            primer strand portion;    -   e) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the partner nucleotide for the next nucleotide of the        predefined sequence in that cycle is the penultimate nucleotide        of the support strand and occupies position n, and the universal        nucleotide occupies position n+2 in the support strand and is        paired with the penultimate nucleotide of the helper strand;        wherein position n is the nucleotide position which upon        ligation of the ligation polynucleotide to the cleaved scaffold        polynucleotide will be opposite the next nucleotide of the        predefined sequence incorporated in that cycle (step 7), and        wherein position n+2 is the second nucleotide position in the        support strand relative to position n in the direction proximal        to the helper strand/distal to the primer strand portion.

Any such method may be performed according to a method of the inventionwhich is a variation of synthesis method version 5 wherein:

-   -   (i) in the cleavage step of the first cycle (step 2) the        universal nucleotide instead occupies position n+2 in the        support strand of the scaffold polynucleotide, wherein n+2 is        the second nucleotide position in the support strand relative to        position n in the direction proximal to the helper strand/distal        to the primer strand portion; and the support strand of the        scaffold polynucleotide is cleaved between positions n and n−1;    -   (ii) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the universal nucleotide instead occupies        position n+3 in the support strand and is paired with the        nucleotide which is 2 positions removed from the terminal        nucleotide of the helper strand at the complementary ligation        end; wherein position n+3 is position 3 in the support strand        relative to position n in the direction proximal to the helper        strand/distal to the primer strand portion;    -   (iii) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles the universal nucleotide        occupies position n+2 in the support strand of the scaffold        polynucleotide, and the support strand of the scaffold        polynucleotide is cleaved between positions n and n−1; and    -   (iv) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the universal nucleotide occupies position n+3 in the        support strand and is paired with the nucleotide which is 2        positions removed from the terminal nucleotide of the helper        strand at the complementary ligation end.

Any such method may be performed according to a method of the inventionwhich is a further variation of synthesis method version 5 wherein:

-   -   (i) in the cleavage step of the first cycle (step 2) the        universal nucleotide instead occupies position n+2+x in the        support strand of the scaffold polynucleotide, wherein n+2 is        the second nucleotide position in the support strand relative to        position n in the direction proximal to the helper strand/distal        to the primer strand portion; and the support strand of the        scaffold polynucleotide is cleaved between positions n and n−1;    -   (ii) in the ligation step of the first cycle (step 5) the        complementary ligation end of the ligation polynucleotide is        structured such that the universal nucleotide instead occupies        position n+3+x in the support strand and is paired with the        nucleotide which is 2+x positions removed from the terminal        nucleotide of the helper strand at the complementary ligation        end; wherein position n+3 is position 3 in the support strand        relative to position n in the direction proximal to the helper        strand/distal to the primer strand portion;    -   (iii) in the cleavage step of the second cycle (step 6) and in        cleavage steps of all subsequent cycles the universal nucleotide        occupies position n+2+x in the support strand of the scaffold        polynucleotide and the support strand of the scaffold        polynucleotide is cleaved between positions n and n−1;    -   (iv) in the ligation step of the second cycle (step 9) and in        ligation steps of all subsequent cycles the complementary        ligation end of the ligation polynucleotide is structured such        that the universal nucleotide occupies position n+3+x in the        support strand and is paired with the nucleotide which is 2+x        positions removed from the terminal nucleotide of the helper        strand at the complementary ligation end; and    -   (v) wherein x is a whole number between 1 to 10 or more, and        wherein x is the same whole number in steps (2), (5), (6) and        (9).

In any of the methods described above and herein, a partner nucleotidewhich pairs with a first/next nucleotide of the predefined sequence maybe a nucleotide which is complementary with the first/next nucleotide,preferably naturally complementary.

In any of the methods described above and herein, in any one or morecycles of synthesis, or in all cycles of synthesis, prior to step (2)and/or (6) the scaffold polynucleotide may be provided comprising asynthesis strand and a support strand hybridized thereto, and whereinthe synthesis strand is provided without a helper strand. In any one ormore cycles of synthesis, or in all cycles of synthesis, prior to step(2) and/or (6) the synthesis strand may be removed from the scaffoldpolynucleotide.

In any of the methods described above and herein, in any one or morecycles of synthesis, or in all cycles of synthesis, after the step ofligating the double-stranded ligation polynucleotide to the cleavedscaffold polynucleotide and before the step of incorporating the nextnucleotide of the predefined nucleotide sequence into the synthesisstrand of the scaffold polynucleotide, the helper strand portion of thesynthesis strand may be removed from the scaffold polynucleotide. In anysuch method the helper strand portion of the synthesis strand may beremoved from the scaffold polynucleotide by: (i) heating the scaffoldpolynucleotide to a temperature of about 80° C. to about 95° C. andseparating the helper strand portion from the scaffold polynucleotide,(ii) treating the scaffold polynucleotide with urea solution, such as 8Murea and separating the helper strand portion from the scaffoldpolynucleotide, (iii) treating the scaffold polynucleotide withformamide or formamide solution, such as 100% formamide and separatingthe helper strand portion from the scaffold polynucleotide, or (iv)contacting the scaffold polynucleotide with a single-strandedpolynucleotide molecule which comprises a region of nucleotide sequencewhich is complementary with the sequence of the helper strand portion,thereby competitively inhibiting the hybridisation of the helper strandportion to the scaffold polynucleotide.

In any such method described above and herein wherein the universalnucleotide occupies position n and wherein the support strand of thescaffold polynucleotide is cleaved between positions n and n−1, eachcleavage step may comprise a two-step cleavage process wherein eachcleavage step may comprise a first step comprising removing theuniversal nucleotide thus forming an abasic site, and a second stepcomprising cleaving the support strand at the abasic site. In any suchmethod the first step may be performed with a nucleotide-excisingenzyme. The nucleotide-excising enzyme may be a 3-methyladenine DNAglycosylase enzyme. The nucleotide-excising enzyme may be humanalkyladenine DNA glycosylase (hAAG) or uracil DNA glycosylase (UDG). Inany such method the second step may be performed with a chemical whichis a base. The base may be NaOH. In any such method the second step maybe performed with an organic chemical having abasic site cleavageactivity. The organic chemical may be N,N′-dimethylethylenediamine. Inany such method the second step may be performed with an enzyme havingabasic site lyase activity such as AP Endonuclease, Endonuclease III(Nth) or Endonuclease VIII.

In any such method described above and herein wherein the universalnucleotide occupies position n and wherein the support strand of thescaffold polynucleotide is cleaved between positions n and n−1, eachcleavage step may comprise a one step cleavage process comprisingremoving the universal nucleotide with a cleavage enzyme, wherein theenzyme is Endonuclease III, Endonuclease VIII, formamidopirimidine DNAglycosylase (Fpg) or 8-oxoguanine DNA glycosylase (hOGG1).

In any such method described above and herein wherein the universalnucleotide occupies position n+1 and wherein the support strand of thescaffold polynucleotide is cleaved between positions n and n−1, or inany such method described above and herein wherein the universalnucleotide occupies position n and wherein the support strand of thescaffold polynucleotide is cleaved between positions n−1 and n−2, thecleavage step may comprise cleaving the support strand with an enzyme.The enzyme may cleave the support strand after the nucleotide which isnext to the universal nucleotide in the direction proximal to the primerstrand portion, thereby creating the overhanging end in the synthesisstrand comprising the first/next nucleotide. Such an enzyme may beEndonuclease V.

In any of the methods described above and herein, both strands of thesynthesised double-stranded polynucleotide may be DNA strands. Thesynthesis strand and the support strand may be DNA strands. In suchcases incorporated nucleotides are preferably dNTPs, preferably dNTPscomprising a reversible terminator group. In any such method any one ormore or all of the incorporated nucleotides comprising a reversibleterminator group may comprise 3′-O-allyl-dNTPs or3′-O-azidomethyl-dNTPs.

In any of the methods described above and herein, a first strand of thesynthesised double-stranded polynucleotide may be a DNA strand and thesecond strand of the synthesised double-stranded polynucleotide may bean RNA strand. The synthesis strand may be an RNA strand and the supportstrand may be a DNA strand. In such cases incorporated nucleotides arepreferably NTPs, preferably NTPs comprising a reversible terminatorgroup. In any such method any one or more or all of the incorporatednucleotides comprising a reversible terminator group may be3′-O-allyl-NTPs or 3′-O-azidomethyl-NTPs.

In any of the methods described above and herein involving incorporationof a nucleotide into a synthesis strand comprising DNA e.g.incorporation of one or more dNTPs, the enzyme may be a polymerase,preferably a DNA polymerase, more preferably a modified DNA polymerasehaving an enhanced ability to incorporate a dNTP comprising a reversibleterminator group compared to an unmodified polymerase. The polymerasemay be a variant of the native DNA polymerase from Thermococcus species9° N, preferably species 9° N-7.

In any of the methods described above and herein involving incorporationof a nucleotide into a synthesis strand comprising RNA e.g.incorporation of one or more NTPs, the enzyme may be a polymerase,preferably an RNA polymerase such as T3 or T7 RNA polymerase, morepreferably a modified RNA polymerase having an enhanced ability toincorporate an NTP comprising a reversible terminator group compared toan unmodified polymerase.

In any of the methods described above and herein, a first strand of thesynthesised double-stranded polynucleotide may be a DNA strand and thesecond strand of the synthesised double-stranded polynucleotide may bean RNA strand. Alternatively, a first strand of the synthesiseddouble-stranded polynucleotide may be an RNA strand and the secondstrand of the synthesised double-stranded polynucleotide may be a DNAstrand.

In any of the methods described above and herein, the enzyme enzyme hasa terminal transferase activity, optionally wherein the enzyme is aterminal nucleotidyl transferase, a terminal deoxynucleotidyltransferase, terminal deoxynucleotidyl transferase (TdT), pol lambda,pol micro or 129 DNA polymerase.

In any of the methods described above and herein, the step of removingthe reversible terminator group from the first/next nucleotide may beperformed with tris(carboxyethyl)phosphine (TCEP).

In any of the methods described above and herein, the step of ligating adouble-stranded ligation polynucleotide to the cleaved scaffoldpolynucleotide is preferably performed with a ligase enzyme. The ligaseenzyme may be a T3 DNA ligase or a T4 DNA ligase.

In any of the methods described above and herein, in step (1), (5)and/or (9) the helper strand and the portion of the support strandhybridized thereto may be connected by a hairpin loop.

In any of the methods described above and herein, in step (1) thesynthesis strand comprising the primer strand portion and the portion ofthe support strand hybridized thereto may be connected by a hairpinloop.

In any of the methods described above and herein, in step (1), (5)and/or (9):

-   -   a) the helper strand and the portion of the support strand        hybridized thereto may be connected by a hairpin loop; and    -   b) the synthesis strand comprising the primer strand portion and        the portion of the support strand hybridized thereto may be        connected by a hairpin loop.

In any of the methods described above and herein, at least one or moreor all of the ligation polynucleotides may be provided as a singlemolecule comprising a hairpin loop connecting the support strand and thehelper strand at the end opposite the complementary ligation end. In anyof the methods described above and herein, the ligation polynucleotidesof each synthesis cycle may be provided as single molecules eachcomprising a hairpin loop connecting the support strand and the helperstrand at the end opposite the complementary ligation end.

In any of the methods described above and herein, in step (1) thesynthesis strand comprising the primer strand portion and the portion ofthe support strand hybridized thereto may be tethered to a commonsurface. The primer strand portion and the portion of the support strandhybridized thereto may each comprise a cleavable linker, wherein thelinkers may be cleaved to detach the double-stranded polynucleotide fromthe surface following synthesis.

In any of the methods described above and herein, in step (1) the primerstrand portion of the synthesis strand and the portion of the supportstrand hybridized thereto may be connected by a hairpin loop, andwherein the hairpin loop is tethered to a surface.

In any of the methods described above and herein, a hairpin loop may betethered to a surface via a cleavable linker, wherein the linker may becleaved to detach the double-stranded polynucleotide from the surfacefollowing synthesis. The cleavable linker may be a UV cleavable linker.

In any of the methods described above and herein, the surface to whichpolynucleotides are attached may be the surface of a microparticle or aplanar surface.

In any of the methods described above and herein, the surface to whichpolynucleotides are attached may comprise a gel. The surface comprises apolyacrylamide surface, such as about 2% polyacrylamide, preferablywherein the polyacrylamide surface is coupled to a solid support such asglass.

In any of the methods described above and herein, the synthesis strandcomprising the primer strand portion and the portion of the supportstrand hybridized thereto may tethered to the common surface via one ormore covalent bonds. The one or more covalent bonds may be formedbetween a functional group on the common surface and a functional groupon the scaffold molecule, wherein the functional group on the scaffoldmolecule may be an amine group, a thiol group, a thiophosphate group ora thioamide group. The functional group on the common surface may be abromoacetyl group, optionally wherein the bromoacetyl group is providedon a polyacrylamide surface derived using N-(5-bromoacetamidylpentyl)acrylamide (BRAPA).

In any of the methods described above and herein, the step of removingthe reversible terminator group from a nucleotide of the predefinedsequence may be performed before the cleavage step, or before theligation step.

In any of the methods described above and herein, reactions relating toany of the synthesis cycles described above and herein may be performedin droplets within a microfluidic system. The microfluidic system may bean electrowetting system. The microfluidic system may be anelectrowetting-on-dielectric system (EWOD).

In any of the methods described above and herein, following synthesisthe strands of the double-stranded polynucleotides may be separated toprovide a single-stranded polynucleotide having a predefined sequence.

In any of the methods described above and herein, following synthesisthe double-stranded polynucleotide or a region thereof is amplified,preferably by PCR.

The invention also provides a method of assembling a polynucleotidehaving a predefined sequence, the method comprising performing any ofthe synthesis methods described above and herein to synthesise a firstpolynucleotide having a predefined sequence and one or more additionalpolynucleotides having a predefined sequence and joining together thefirst and the one or more additional polynucleotides. The first and theone or more additional polynucleotides may preferably comprise differentpredefined sequences. The first polynucleotide and the one or moreadditional polynucleotides may be double-stranded or may besingle-stranded. The first polynucleotide and the one or more additionalpolynucleotides may first be cleaved to create compatible termini andthen joined together, e.g. by ligation. The first polynucleotide and theone or more additional polynucleotides may be cleaved by a restrictionenzyme at a cleavage site to create compatible termini.

Any of the in vitro methods for synthesising a double-strandedpolynucleotide having a predefined sequence as described above andherein, and/or any of the in vitro methods of assembling apolynucleotide having a predefined sequence as described above andherein may be performed in droplets within a microfluidic system. In anysuch methods, the assembly methods may comprise assembly steps whichcomprise providing a first droplet comprising a first synthesisedpolynucleotide having a predefined sequence and a second dropletcomprising an additional one or more synthesised polynucleotides havinga predefined sequence, wherein the droplets are brought in contact witheach other and wherein the synthesised polynucleotides are joinedtogether thereby assembling a polynucleotide comprising the first andadditional one or more polynucleotides. In any such methods thesynthesis steps may be performed by providing a plurality of dropletseach droplet comprising reaction reagents corresponding to a step of thesynthesis cycle, and sequentially delivering the droplets to thescaffold polynucleotide in accordance with the steps of the synthesiscycles. In any such methods, following delivery of a droplet and priorto the delivery of a next droplet, a washing step may be carried out toremove excess reaction reagents. In any such methods the microfluidicsystem may be an electrowetting system. In any such methods themicrofluidic system may be an electrowetting-on-dielectric system(EWOD). In any such methods the synthesis and assembly steps may beperformed within the same system.

In a related aspect, the invention further provides the use of auniversal nucleotide in an in vitro method of synthesising adouble-stranded polynucleotide having a predefined sequence, wherein theuniversal nucleotide is used to create a polynucleotide cleavage siteduring each cycle of synthesis, wherein in each synthesis cycle said usecomprises: providing a scaffold polynucleotide comprising a synthesisstrand and a support strand hybridized thereto, wherein the synthesisstrand comprises a primer strand portion and optionally a helper strandportion separated from the primer strand portion by a single-strandbreak, and wherein the universal nucleotide is provided in the supportstrand to provide a cleavage site; cleaving the scaffold polynucleotideat the cleavage site whereupon the universal nucleotide is removed fromthe scaffold polynucleotide and a cleaved end is created in the scaffoldpolynucleotide; adding to the terminal end of the synthesis strand atthe cleaved end of the scaffold polynucleotide by a polymerase or atransferase enzyme a new nucleotide of the predefined sequencecomprising a reversible terminator group; ligating to the cleaved end ofthe scaffold polynucleotide a ligation polynucleotide having a supportstrand and a helper strand hybridized thereto, the support strandcomprising a nucleotide for pairing with the new nucleotide in thesynthesis strand of the scaffold polynucleotide and further comprising anew universal nucleotide for use in creating a new polynucleotidecleavage site for use in the next cycle of synthesis, wherein thereversible terminator group is removed from the new nucleotide after thecleavage or ligation step; and optionally wherein the helper strand isremoved after the ligation step and before the cleavage step of the nextcycle. Such use of a universal nucleotide in a method of synthesising adouble-stranded polynucleotide having a predefined sequence may beimplemented using any of the specific methods defined and describedabove and herein.

In a related aspect, the invention further provides an in vitro methodof extending a synthesis strand of a polynucleotide molecule with apredefined nucleotide, the method comprising: providing a scaffoldpolynucleotide comprising the synthesis strand and a support strandhybridized thereto, wherein the synthesis strand comprises a primerstrand portion and optionally a helper strand portion separated from theprimer strand portion by a single-strand break, and wherein theuniversal nucleotide is provided in the support strand and defines apolynucleotide cleavage site; cleaving the scaffold polynucleotide atthe cleavage site whereupon the universal nucleotide is removed from thescaffold polynucleotide and a cleaved end is created in the scaffoldpolynucleotide; adding to the terminal end of the synthesis strand atthe cleaved end of the scaffold polynucleotide by a polymerase or atransferase enzyme a new nucleotide of the predefined sequencecomprising a reversible terminator group; wherein the cleaved end of thescaffold polynucleotide acts as a ligation acceptor site for a ligationpolynucleotide having a support strand and a helper strand hybridizedthereto, the support strand comprising a nucleotide for pairing with thepredefined nucleotide in the synthesis strand of the scaffoldpolynucleotide and further comprising a new universal nucleotide for usein creating a new polynucleotide cleavage site for use in the next cycleof synthesis, wherein the reversible terminator group is removed fromthe new nucleotide after the cleavage or ligation step and optionallywherein the helper strand is removed after the ligation step and beforethe cleavage step of the next cycle. In any such method of extending asynthesis strand of a polynucleotide molecule with a predefinednucleotide, the method may be implemented using any of the specificmethods defined and described above and herein.

In a related aspect, the invention further provides an in vitro methodof synthesising a double-stranded polynucleotide having a predefinedsequence, the method comprising cycles of synthesis and wherein eachsynthesis cycle comprises: providing a scaffold polynucleotidecomprising a synthesis strand and a support strand hybridized thereto,wherein the synthesis strand comprises a primer strand portion andoptionally a helper strand portion separated from the primer strandportion by a single-strand break, and wherein a universal nucleotide isprovided in the support strand and defines a polynucleotide cleavagesite; cleaving the scaffold polynucleotide at the cleavage sitewhereupon the universal nucleotide is removed from the scaffoldpolynucleotide and a cleaved end is created in the scaffoldpolynucleotide; adding to the terminal end of the synthesis strand atthe cleaved end of the scaffold polynucleotide by a polymerase or atransferase enzyme a new nucleotide of the predefined sequencecomprising a reversible terminator group; ligating to the cleaved end ofthe scaffold polynucleotide a ligation polynucleotide having a supportstrand and a helper strand hybridized thereto, the support strandcomprising a nucleotide for pairing with the new nucleotide in thesynthesis strand of the scaffold polynucleotide and further comprising anew universal nucleotide for use in creating a new polynucleotidecleavage site for use in the next cycle of synthesis; removing thereversible terminator group from the new nucleotide after the cleavageor ligation step; and optionally removing the helper strand after theligation step and before the cleavage step of the next cycle. In anysuch method of synthesising a double-stranded polynucleotide having apredefined sequence, the method may be implemented using any of thespecific methods defined and described above and herein.

In a related aspect, the invention further provides an in vitro methodof ligating a ligation polynucleotide comprising a universal nucleotideto a double-stranded polynucleotide during a cycle of synthesising adouble-stranded polynucleotide having a predefined sequence, whereinduring the synthesis cycle the double-stranded polynucleotide isextended with a predefined nucleotide and a partner therefor; the methodcomprising providing a scaffold polynucleotide comprising a synthesisstrand and a support strand hybridized thereto, wherein the synthesisstrand comprises a primer strand portion and optionally a helper strandportion separated from the primer strand portion by a single-strandbreak, and wherein a universal nucleotide is provided in the supportstrand and defines a polynucleotide cleavage site; cleaving the scaffoldpolynucleotide at the cleavage site whereupon the universal nucleotideis removed from the scaffold polynucleotide and a cleaved end is createdin the scaffold polynucleotide; adding to the terminal end of thesynthesis strand at the cleaved end of the scaffold polynucleotide by apolymerase or a transferase enzyme a new nucleotide of the predefinedsequence comprising a reversible terminator group ligating to thecleaved end of the scaffold polynucleotide a ligation polynucleotidehaving a support strand and a helper strand hybridized thereto, thesupport strand comprising a nucleotide for pairing with the newnucleotide in the synthesis strand of the scaffold polynucleotide andfurther comprising a new universal nucleotide for use in creating a newpolynucleotide cleavage site for use in the next cycle of synthesis,thereby creating a new scaffold polynucleotide for use in the nextsynthesis cycle; removing the reversible terminator group from the newnucleotide after the cleavage or ligation step; and optionally removingthe helper strand after the ligation step and before the cleavage stepof the next cycle. In any such method of ligating a ligationpolynucleotide comprising a universal nucleotide to a double-strandedpolynucleotide during a cycle of synthesising a double-strandedpolynucleotide having a predefined sequence, the method may beimplemented using any of the specific methods defined and describedabove and herein.

The invention additionally provides a polynucleotide synthesis systemfor carrying out any of the synthesis and/or assembly methods describedabove and herein, comprising (a) an array of reaction areas, whereineach reaction area comprises at least one scaffold polynucleotide; and(b) means for the delivery of the reaction reagents to the reactionareas and optionally, (c) means to cleave the synthesiseddouble-stranded polynucleotide from the scaffold polynucleotide. Such asystem may further comprise means for providing the reaction reagents indroplets and means for delivering the droplets to the scaffoldpolynucleotide in accordance with the synthesis cycles.

The invention further provides a kit for use with any of the systemsdescribed above and herein, and for carrying out any of the synthesismethods described above and herein, the kit comprising volumes ofreaction reagents corresponding to the steps of the synthesis cycles.

The invention also provides a method of making a polynucleotidemicroarray, wherein the microarray comprises a plurality of reactionareas, each area comprising one or more polynucleotides having apredefined sequence, the method comprising:

-   -   a) providing a surface comprising a plurality of reaction areas,        each area comprising one or more double-stranded anchor or        scaffold polynucleotides, and    -   b) performing cycles of synthesis according to any of the        methods described above and herein at each reaction area,        thereby synthesising at each area one or more double-stranded        polynucleotides having a predefined sequence.

In such methods, following synthesis the strands of the double-strandedpolynucleotides may be separated to provide a microarray wherein eacharea comprises one or more single-stranded polynucleotides having apredefined sequence.

DESCRIPTION OF THE FIGURES

Relevant Figures presented herein and described below show some or allof the steps of a cycle of synthesis using methods, including methods ofthe invention, as well as means for performing aspects of the methods,such as oligonucleotides, surfaces, surface attachment chemistries,linkers etc. These Figures as well as all descriptions thereof and allassociated methods, reagents and protocols are presented forillustration only and are not to be construed as limiting.

Relevant Figures, such as e.g. FIGS. 6, 7, 8, 9, 10, 13 a, 14 a, 15 aetc. show some or all of the steps of a cycle of synthesis includingincorporation of a nucleotide (e.g., a nucleotide comprising areversible terminator group), cleavage (e.g., cleaving the scaffoldpolynucleotide into a first portion and a second portion, wherein thefirst portion comprises an universal nucleotide, and the second portioncomprises the incorporated nucleotide), ligation (e.g., ligating to thesecond portion of the cleaved scaffold polynucleotide comprising theincorporated nucleotide, a polynucleotide construct comprising asingle-stranded portion, wherein the single-stranded portion comprises apartner nucleotide that is complementary to the incorporated nucleotide)and deprotection (e.g., removing the reversible terminator group fromthe incorporated nucleotide). These methods are provided forillustrative support only and are not within the scope of the claimedinvention. Method schemes shown in FIGS. 1 to 5 are methods of theinvention. The data shown in FIG. 51, as described in Example 13, relateto methods of the invention consistent with synthesis method versions 1,2 and 4 as depicted in FIGS. 1, 2 and 4 as described herein.

FIG. 1. Scheme of Exemplary Method Version 1 of the Invention.

Scheme showing a first synthesis cycle according to exemplary methodversion 1 of the invention. The method comprises a cycle of provision ofa scaffold polynucleotide, cleavage, incorporation, deprotection andligation. The scheme shows the provision of a scaffold polynucleotide(101) comprising a universal nucleotide in the support strand whichdefines a polynucleotide cleavage site and a single-stranded break(“nick”) in the synthesis strand. The scheme shows cleavage of thesupport strand by the creation of a single-stranded break in the supportstrand (102) followed by addition of a thymine nucleotide to theterminal end of the synthesis strand at the cleaved end of the scaffoldpolynucleotide (103) and removal of the reversible blocking group duringa protection step (104). Following ligation of a ligation polynucleotideto the cleaved scaffold polynucleotide (105) the newly incorporatedthymine nucleotide is paired opposite its partner nucleotide adenine(104). Following the ligation step a reconstituted scaffoldpolynucleotide is provided with the newly incorporated pair ofnucleotides (106) for use in the next synthesis cycle. This A-T pair isshown for illustration purposes only and is not limiting, it can be anypair depending on the required predefined sequence. Nucleotide pairs atpositions H-I can be any pair. Nucleotide X can be any nucleotide. TheFigure also shows reference signs corresponding to a second synthesiscycle.

FIG. 2. Scheme of Exemplary Method Version 2 of the Invention.

Scheme showing a first synthesis cycle according to exemplary methodversion 2 of the invention. The method comprises a cycle of provision ofa scaffold polynucleotide, cleavage, incorporation, deprotection andligation. The scheme shows the provision of a scaffold polynucleotide(101) comprising a universal nucleotide in the support strand whichdefines a polynucleotide cleavage site and a single-stranded break(“nick”) in the synthesis strand. The scheme shows cleavage of thesupport strand by the creation of a single-stranded break in the supportstrand (102) followed by addition of a thymine nucleotide to theterminal end of the synthesis strand at the cleaved end of the scaffoldpolynucleotide (103) and removal of the reversible blocking group duringa protection step (104). Following ligation of a ligation polynucleotideto the cleaved scaffold polynucleotide (105) the newly incorporatedthymine nucleotide is paired opposite its partner nucleotide adenine(104). Following the ligation step a reconstituted scaffoldpolynucleotide is provided with the newly incorporated pair ofnucleotides (106) for use in the next synthesis cycle. This A-T pair isshown for illustration purposes only and is not limiting, it can be anypair depending on the required predefined sequence. Nucleotide pairs atpositions H-I and J-K can be any pair. Nucleotide X can be anynucleotide. The Figure also shows reference signs corresponding to asecond synthesis cycle.

FIG. 3. Scheme of Exemplary Method Version 3 of the Invention.

Scheme showing a first synthesis cycle according to exemplary methodversion 3 of the invention. The method comprises a cycle of provision ofa scaffold polynucleotide, cleavage, incorporation, deprotection andligation. The scheme shows the provision of a scaffold polynucleotide(101) comprising a universal nucleotide in the support strand whichdefines a polynucleotide cleavage site and a single-stranded break(“nick”) in the synthesis strand. The scheme shows cleavage of thesupport strand by the creation of a single-stranded break in the supportstrand (102) followed by addition of a thymine nucleotide to theterminal end of the synthesis strand at the cleaved end of the scaffoldpolynucleotide (103) and removal of the reversible blocking group duringa protection step (104). Following ligation of a ligation polynucleotideto the cleaved scaffold polynucleotide (105) the newly incorporatedthymine nucleotide is paired opposite its partner nucleotide adenine(104). Following the ligation step a reconstituted scaffoldpolynucleotide is provided with the newly incorporated pair ofnucleotides (106) for use in the next synthesis cycle. This A-T pair isshown for illustration purposes only and is not limiting, it can be anypair depending on the required predefined sequence. Nucleotide pairs atpositions H-I and J-K can be any pair. Nucleotide X can be anynucleotide. The Figure also shows reference signs corresponding to asecond synthesis cycle.

FIG. 4. Scheme of Exemplary Method Version 4 of the Invention.

Scheme showing a first synthesis cycle according to exemplary methodversion 4 of the invention. The method comprises a cycle of provision ofa scaffold polynucleotide, cleavage, incorporation, deprotection andligation. The scheme shows the provision of a scaffold polynucleotide(101) comprising a universal nucleotide in the support strand whichdefines a polynucleotide cleavage site and a single-stranded break(“nick”) in the synthesis strand. The scheme shows cleavage of thesupport strand by the creation of a single-stranded break in the supportstrand (102) followed by addition of a thymine nucleotide to theterminal end of the synthesis strand at the cleaved end of the scaffoldpolynucleotide (103) and removal of the reversible blocking group duringa protection step (104). Following ligation of a ligation polynucleotideto the cleaved scaffold polynucleotide (105) the newly incorporatedthymine nucleotide is paired opposite its partner nucleotide adenine(104). Following the ligation step a reconstituted scaffoldpolynucleotide is provided with the newly incorporated pair ofnucleotides (106) for use in the next synthesis cycle. This A-T pair isshown for illustration purposes only and is not limiting, it can be anypair depending on the required predefined sequence. Nucleotide pairs atpositions H-I, J-K and L-M can be any pair. Nucleotide X can be anynucleotide. The Figure also shows reference signs corresponding to asecond synthesis cycle.

FIG. 5. Scheme of Exemplary Method Version 5 of the Invention.

The scheme shows the provision of a scaffold polynucleotide (101)comprising a universal nucleotide in the support strand which defines apolynucleotide cleavage site and a single-stranded break (“nick”) in thesynthesis strand. The scheme shows cleavage of the support strand by thecreation of a single-stranded break in the support strand (102) followedby addition of a thymine nucleotide to the terminal end of the synthesisstrand at the cleaved end of the scaffold polynucleotide (103) andremoval of the reversible blocking group during a protection step (104).Following ligation of a ligation polynucleotide to the cleaved scaffoldpolynucleotide (105) the newly incorporated thymine nucleotide is pairedopposite its partner nucleotide adenine (104). Following the ligationstep a reconstituted scaffold polynucleotide is provided with the newlyincorporated pair of nucleotides (106) for use in the next synthesiscycle. This A-T pair is shown for illustration purposes only and is notlimiting, it can be any pair depending on the required predefinedsequence. Nucleotide pairs at positions H-I, J-K and L-M can be anypair. Nucleotide X can be any nucleotide. The Figure also showsreference signs corresponding to a second synthesis cycle.

FIG. 6. Scheme of Exemplary Method Version 1.

Scheme showing a first synthesis cycle according to exemplary methodversion 1 of the Examples section. This method is provided forillustrative support only and is not within the scope of the claimedinvention. The method comprises a cycle of provision of a scaffoldpolynucleotide, incorporation, cleavage, ligation and deprotection. Thescheme shows the incorporation of a thymine nucleotide in the firstsynthesis cycle (101, 102) and its pairing opposite a partner adeninenucleotide (104), as well as the provision of a scaffold polynucleotide(106) for use in the next synthesis cycle. This pair is shown forillustration purposes only and is not limiting, it can be any pairdepending on the required predefined sequence. Nucleotide Z can be anynucleotide. Nucleotide X can be any appropriate nucleotide. The Figurealso shows reference signs corresponding to a second synthesis cycle.

FIG. 7. Scheme of Exemplary Method Version 2.

Scheme showing a first synthesis cycle according to exemplary methodversion 2 of the Examples section. This method is provided forillustrative support only and is not within the scope of the claimedinvention. The method comprises a cycle of provision of a scaffoldpolynucleotide, incorporation, cleavage, ligation and deprotection. Thescheme shows the incorporation in the first cycle (201, 202) of athymine nucleotide and its pairing opposite a partner adenine nucleotide(204), as well as the provision of a scaffold polynucleotide (206)comprising a guanine for pairing with a cytosine in the next synthesiscycle. These pairs are shown for illustration purposes only and are notlimiting, they can be any pairs depending on the required predefinedsequence. Nucleotide Z can be any nucleotide. Nucleotide X can be anyappropriate nucleotide. The Figure also shows reference signscorresponding to a second synthesis cycle.

FIG. 8. Scheme of Exemplary Method Version 3.

Scheme showing a first synthesis cycle according to exemplary methodversion 3 of the Examples section. This method is provided forillustrative support only and is not within the scope of the claimedinvention. The method comprises a cycle of provision of a scaffoldpolynucleotide, incorporation, cleavage, ligation and deprotection. Thescheme shows the incorporation in the first cycle (301, 302) of athymine nucleotide and its pairing opposite a partner adenine nucleotide(304), as well as the provision of a scaffold polynucleotide (306) foruse in the next synthesis cycle. This pair is shown for illustrationpurposes only and is not limiting, it can be any pair depending on therequired predefined sequence. The scheme also shows a cytosine-guaninepair as a component of the scaffold polynucleotide and which is not partof the predefined sequence. This pair is also shown for illustrationpurposes only and is not limiting, it can be any pair. Nucleotide Z canbe any nucleotide. Nucleotide X can be any appropriate nucleotide.

FIG. 9. Scheme of Exemplary Method Version 4.

Scheme showing a first synthesis cycle according to exemplary methodversion 4 of the Examples section. This method is provided forillustrative support only and is not within the scope of the claimedinvention. The method comprises a cycle of provision of a scaffoldpolynucleotide, incorporation, cleavage, ligation and deprotection. Thescheme shows the incorporation in the first cycle (401, 402) of athymine nucleotide and its pairing opposite a partner universalnucleotide (404), as well as the provision of a scaffold polynucleotide(406) comprising a guanine for pairing with a cytosine in the nextsynthesis cycle. These pairs are shown for illustration purposes onlyand are not limiting, they can be any pairs depending on the requiredpredefined sequence. Nucleotides X, Y and Z can be any nucleotide.

FIG. 10. Scheme of Exemplary Method Version 5.

Scheme showing a first synthesis cycle according to exemplary methodversion 5 of the Examples section. This method is provided forillustrative support only and is not within the scope of the claimedinvention. The method comprises a cycle of provision of a scaffoldpolynucleotide, incorporation, cleavage, ligation and deprotection. Thescheme shows the incorporation in the first cycle (501, 502) of athymine nucleotide and its pairing opposite a partner adenine nucleotide(504), as well as the provision of a scaffold polynucleotide (506)comprising a guanine for pairing with a cytosine in the next synthesiscycle. The scheme also shows a cytosine-guanine pair (position n−2) as acomponent of the scaffold polynucleotide and which is not part of thepredefined sequence. These pairs are shown for illustration purposesonly and are not limiting, they can be any pairs depending on therequired predefined sequence. Nucleotides X, Y and Z can be anynucleotide.

FIG. 11. Scheme Showing Surface Immobilization of ScaffoldPolynucleotides.

Schemes show (a to h) possible example hairpin loop configurations ofscaffold polynucleotides and their immobilisation to surfaces.

Schemes (i and j) show examples of surface chemistries for attachingpolynucleotides to surfaces. The examples show double-strandedembodiments wherein both strands are connected via a hairpin, but thesame chemistries may be used for attaching one or both strands of anunconnected double-stranded polynucleotide.

FIG. 12. Absence of Helper Strand—Incorporation.

a) Scheme showing incorporation step highlighted in dashed box.

b) Evaluation of DNA polymerases for incorporation of3′-O-modified-dTTPs opposite inosine. The Figure depicts a gel showingresults of incorporation of 3′-O-modified-dTTPs by various DNApolymerases (Bst, Deep Vent (Exo-), Therminator I and Therminator IX) inpresence of Mn²⁺ ions at 50° C. Lane 1: Incorporation of3′-O-allyl-dTTPs using Bst DNA polymerase. Lane 2: Incorporation of3′-O-azidomethyl-dTTPs using Bst DNA polymerase. Lane 3: Incorporationof 3′-O-allyl-dTTPs using Deep vent (exo-) DNA polymerase. Lane 4:Incorporation of 3′-O-azidomethyl-dTTPs using Deep vent (exo-) DNApolymerase. Lane 5: Incorporation of 3′-O-allyl-dTTPs using TherminatorI DNA polymerase. Lane 6: Incorporation of 3′-O-azidomethyl-dTTPs usingTherminator I DNA polymerase. Lane 7: Incorporation of 3′-O-allyl-dTTPsusing Therminator IX DNA polymerase. Lane 8: Incorporation of3′-O-azidomethyl-dTTPs using Therminator IX DNA polymerase.

c) Evaluation of DNA polymerases for incorporation of3′-O-modified-dTTPs opposite inosine. Results of incorporation usingvarious DNA polymerases.

d) Evaluation of the temperature on the incorporation using TherminatorIX DNA polymerase. The Figure depicts a gel showing results ofincorporation of 3′-modified-dTTP opposite inosine in presence of Mn²⁺ions using Therminator IX DNA polymerase at various temperatures. Lane1: Incorporation of 3′-O-allyl-dTTPs at 37° C. Lane 2: Incorporation of3′-O-azidomethyl-dTTPs at 37° C. Lane 3: Incorporation of3′-O-allyl-dTTPs at 50° C. Lane 4: Incorporation of3′-O-azidomethyl-dTTPs at 50° C. Lane 5: Incorporation of3′-O-allyl-dTTPs at 65° C. Lane 6: Incorporation of3′-O-azidomethyl-dTTPs at 65° C.

e) Evaluation of the temperature on the incorporation using TherminatorIX DNA polymerase. Results of incorporation performed at differenttemperatures.

f) Evaluation of the presence of Mn²⁺ on the incorporation usingTherminator IX DNA polymerase. The Figure depicts a gel showing resultsof incorporation of 3′-O-modified-dTTP opposite inosine at 65° C. LaneS: Standards. Lane 1: Incorporation of 3′-O-allyl-dTTPs without Mn²⁺ions. Lane 2: Incorporation of 3′-O-azidomethyl-dTTPs without Mn²⁺ ions.Lane 3: Incorporation of 3′-O-allyl-dTTPs in presence of Mn²⁺ ions. Lane4: Incorporation of 3′-O-azidomethyl-dTTPs in presence of Mn²⁺ ions.

g) Evaluation of the presence of Mn²⁺ on the incorporation usingTherminator IX DNA polymerase. Results of incorporation in presence andabsence of Mn²⁺ ions.

h) Oligonucleotides used for study of the incorporation step.

FIG. 13. Absence of Helper Strand—Cleavage.

a) Scheme showing cleavage of hybridized polynucleotide strands in theabsence of a helper strand. Cleavage step is highlighted in dashed box.

b) Gel showing cleavage of oligonucleotide with hAAG and 0.2M NaOH(strong base) at 37° C. and room temperature 24° C. respectively.Lane 1. Starting oligonucleotide. Lane 2 which was a positive controlthat contained both full length strands showed a higher yield of cleavedto uncleaved DNA ratio of 90%:10%. Lane 3 which included the cleavagereaction without a helper strand showed a low percentage yield ofcleaved to uncleaved DNA ratio of 10%:90%.

c) Gel showing cleavage of oligonucleotide with hAAG and Endo VIII at37° C. Lane 2 which was a positive control that contained both fulllength strands showed a higher yield of cleaved to uncleaved DNA ratioof ˜90%:10%. Lane 3 which included the cleavage reaction without ahelper strand showed a low percentage yield of cleaved to uncleaved DNAratio of ˜7%:93%.

d) A summary of cleavage of oligonucleotide with hAAG/Endo VIII andhAAG/Chemical base.

e) Oligonucleotides used for study of the cleavage step.

FIG. 14. Absence of Helper Strand—Ligation.

a) Scheme showing ligation of hybridized polynucleotide strands in theabsence of a helper strand. Ligation step highlighted in dashed box.

b) Gel showing ligation of Oligonucleotides with Quick T4 DNA ligase atroom temperature (24° C.) in the absence of a helper strand. Lane 1contained a mixture of the 36mers TAMRA single stranded oligos and18mers TAMRA single stranded oligos. These oligos served referencebands.

c) Oligonucleotides used for study of the ligation step.

FIG. 15. Version 1 Chemistry with Helper Strand—Incorporation.

-   -   a) Scheme showing incorporation step highlighted in dashed box.    -   b) Oligonucleotides applicable for study of the incorporation        step.

FIG. 16. Version 1 Chemistry with Helper Strand—Cleavage.

a) Scheme showing cleavage of hybridized polynucleotide strands in theabsence of a helper strand. Cleavage step is highlighted in dashed box.

b) Gel showing cleavage of Oligonucleotide with hAAG and 0.2M NaOH(strong base) at 37° C. and room temperature 24° C. respectively.Lane 1. Starting oligonucleotide. Lane 2 which was a positive controlthat contained both full length strands showed a higher yield of cleavedto uncleaved DNA ratio of 90%:10%. Lane 3 which included the cleavagereaction without a helper strand showed a low percentage yield ofcleaved to uncleaved DNA ratio of 10%:90%. Lane 4 which included thecleavage reaction with a helper strand showed an equal percentage yieldof cleaved to uncleaved DNA ratio of 50%:50%.

c) Evaluation of Endonuclease VIII for cleavage of abasic sites. Gelshows cleavage of oligonucleotide with hAAG and Endo VIII at 37° C. Lane2 which was a positive control that contained both full length strandsshowed a higher yield of cleaved to uncleaved DNA ratio of ˜90%:10%.Lane 3 which included the cleavage reaction without a helper strandshowed a low percentage yield of cleaved to uncleaved DNA ratio of˜7%:93%. Lane 4 which included the cleavage reaction with a helperstrand showed an low percentage yield of cleaved to uncleaved DNA ratioof 10%:90%.

d) Evaluation of N,N′-dimethylethylenediamine for cleavage of abasicsites. Gel shows cleavage of oligonucleotide with hAAG and 100 mMN,N′-dimethylethylenediamine at 37° C. Lane 1. Starting oligonucleotide.Lane 2 which was a positive control that contained both full lengthstrands showed a 100% cleaved DNA. Lane 3 which included the cleavagereaction with a helper strand showed a higher percentage yield ofcleaved to uncleaved DNA ratio of 90%:10%.

e) A summary of cleavage of oligonucleotide with hAAG/Endo VIII,hAAG/chemical base and hAAG/alternative chemical base.

f) Oligonucleotides used for study of the cleavage step.

FIG. 17. Version 1 Chemistry with Helper Strand—Ligation.

a) Scheme showing ligation of hybridized polynucleotide strands in thepresence of a helper strand. Ligation step highlighted in dashed box.

b) Gel showing ligation of oligonucleotides with Quick T4 DNA ligase atroom temperature (24° C.) in the presence of a helper strand. Lane 1contained a mixture of the 36mers TAMRA single stranded oligos and18mers TAMRA single stranded oligos. These oligos served referencebands. In lane 2 there was an observable ligation product of expectedband size 36mers after 20 minutes.

c) Gel showing ligation of oligonucleotides with Quick T4 DNA ligase atroom temperature (24° C.) after overnight incubation in the presence ofa helper strand. Lane 1 contained a mixture of the 36mers TAMRA singlestranded oligos and 18mers TAMRA single stranded oligos. These oligosserved as reference bands. In lane 2 there was an observable completelyligated product of expected band size of 36mers.

d) Oligonucleotides used for study of the ligation step.

FIG. 18. Version 2 Chemistry with Helper Strand—Incorporation.

a) Scheme showing incorporation step highlighted in orange dashed box

b) Gel showing results of incorporation of 3′-O-modified-dTTPs byTherminator IX DNA polymerase at 27° C. Lane 1: Starting material. Lane2: Incorporation after 1 minute, conversion 5%. Lane 3: Incorporationafter 2 minutes, conversion 10%. Lane 4: Incorporation after 5 minutes,conversion 20%. Lane 5: Incorporation after 10 minutes, conversion 30%.Lane 6: Incorporation after 20 minutes, conversion 35%.

c) The Figure depicts a gel showing results of incorporation of3′-O-modified-dTTPs by Therminator IX DNA polymerase at 37° C. Lane 1:Starting material. Lane 2: Incorporation after 1 minute, conversion 30%.Lane 3: Incorporation after 2 minutes, conversion 60%. Lane 4:Incorporation after 5 minutes, conversion 90%. Lane 5: Incorporationafter 10 minutes, conversion 90%. Lane 6: Incorporation after 20minutes, conversion 90%.

d) Gel showing results of incorporation of 3′-O-modified-dTTPs byTherminator IX DNA polymerase at 47° C. Lane 1: Starting material. Lane2: Incorporation after 1 minute, conversion 30%. Lane 3: Incorporationafter 2 minutes, conversion 65%. Lane 4: Incorporation after 5 minutes,conversion 90%. Lane 5: Incorporation after 10 minutes, conversion 90%.Lane 6: Incorporation after 20 minutes, conversion 90%.

e) Gel showing results of incorporation of 3′-O-modified-dTTPs byTherminator IX DNA polymerase at 27° C. Lane 1: Starting material. Lane2: Incorporation after 1 minute, conversion 70%. Lane 3: Incorporationafter 2 minutes, conversion 85%. Lane 4: Incorporation after 5 minutes,conversion 92%. Lane 5: Incorporation after 10 minutes, conversion 96%.Lane 6: Incorporation after 20 minutes, conversion 96%.

f) Gel showing results of incorporation of 3′-O-modified-dTTPs byTherminator IX DNA polymerase at 37° C. Lane 1: Starting material. Lane2: Incorporation after 1 minute, conversion 85%. Lane 3: Incorporationafter 2 minutes, conversion 95%. Lane 4: Incorporation after 5 minutes,conversion 96%. Lane 5: Incorporation after 10 minutes, conversion 96%.Lane 6: Incorporation after 20 minutes, conversion 96%.

g) Gel showing results of incorporation of 3′-O-modified-dTTPs byTherminator IX DNA polymerase at 47° C. Lane 1: Starting material. Lane2: Incorporation after 1 minute, conversion 85%. Lane 3: Incorporationafter 2 minutes, conversion 90%. Lane 4: Incorporation after 5 minutes,conversion 96%. Lane 5: Incorporation after 10 minutes, conversion 96%.Lane 6: Incorporation after 20 minutes, conversion 96%.

h) Summary of incorporation of 3′-O-azidomethyl-dTTP at varioustemperatures and presence of Mn²⁺ ions.

i) Gel showing results of incorporation of 3′-O-modified-dNTPs oppositecomplementary base by Therminator IX DNA polymerase in presence of Mn²⁺at 37° C. Lane 1: Starting material. Lane 2: Incorporation of3′-O-azidomethyl-dTTP for 5 minutes. Lane 3: Incorporation of3′-O-azidomethyl-dATP for 5 minutes. Lane 4: Incorporation of3′-O-azidomethyl-dCTP for 5 minutes. Lane 5: Incorporation of3′-O-azidomethyl-dGTP for 5 minutes.

j) Oligonucleotides used for study of the incorporation step.

FIG. 19. Version 2 Chemistry with Helper Strand—Cleavage.

a) Scheme showing cleavage of hybridized polynucleotide strand in thepresence of a helper strand. Cleavage step is highlighted in orangedashed box.

b) Gel shows cleavage of Oligonucleotide with Endo V at 37° C. Lane 1.Starting oligonucleotide. Lane 2 which was a positive control thatcontained both full length strands showed a yield of cleaved touncleaved DNA ratio of 80%:20%. Lane 3 which included the cleavagereaction without a helper strand showed a much higher yield of cleavedDNA of >99%. Lane 4 which included the cleavage reaction with a helperstrand also showed a DNA cleavage yield of >99%.

c) A summary of cleavage study with Endonuclease V.

d) Oligonucleotides used for study of the cleavage step.

FIG. 20. Version 2 Chemistry with Helper Strand—Ligation.

a) Scheme showing ligation of hybridized polynucleotide strands in theabsence of a helper strand. Ligation step highlighted in orange dashedbox.

b) Oligonucleotides for study of the ligation step.

FIG. 21. Version 2 Chemistry with Helper Strand—Deprotection.

a) Scheme showing deprotection step highlighted in orange dashed box.

b) The Figure depicts a gel showing results of 3′-O-azidomethyl groupdeprotection by 50 mM TCEP after incorporation of 3′-O-azidomethyl-dTTP.Lane 1: Starting primer Lane 2: Incorporation of 3′-O-azidomethyl-dTTPsin presence Mn²±. Lane 3: Extension of the product in lane 2 by additionof all natural dNTPs. Lane 4: Deprotection of the product (0.5 μM) inlane 2 by 50 mM TCEP. Lane 5: Extension of the product in lane 4 byaddition of all natural dNTPs.

c) The Figure depicts a gel showing results of 3′-O-azidomethyl groupdeprotection by 300 mM TCEP after incorporation of3′-O-azidomethyl-dTTP. Lane 1: Starting primer. Lane 2: Incorporation of3-O-azidomethyl-dTTPs in presence Mn²±. Lane 3: Extension of the productin lane 2 by addition of all natural dNTPs. Lane 4: Deprotection of theproduct (0.5 μM) in lane 2 by 300 mM TCEP. Lane 5: Extension of theproduct in lane 4 by addition of all natural dNTPs.

d) The Figure depicts a gel showing results of 3′-O-azidomethyl groupdeprotection by 50 mM TCEP after incorporation of 3′-O-azidomethyl-dCTP.Lane 1: Starting primer. Lane 2: Incorporation of 3-O-azidomethyl-dCTPsin presence Mn²±. Lane 3: Extension of the product in lane 2 by additionof all natural dNTPs. Lane 4: Deprotection of the product (0.5 μM) inlane 2 by 300 mM TCEP. Lane 5: Extension of the product in lane 4 byaddition of all natural dNTPs.

e) The Figure depicts a gel showing results of 3′-O-azidomethyl groupdeprotection by 300 mM TCEP after incorporation of3′-O-azidomethyl-dCTP. Lane 1: Starting primer Lane 2: Incorporation of3-O-azidomethyl-dCTPs in presence Mn²±. Lane 3: Extension of the productin lane 1 by addition of all natural dNTPs. Lane 4: Deprotection of theproduct (0.5 μM) in lane 1 by 300 mM TCEP. Lane 5: Extension of theproduct in lane 3 by addition of all natural dNTPs.

f). The Figure depicts a gel showing results of 3′-O-azidomethyl groupdeprotection by 300 mM TCEP after incorporation of3′-O-azidomethyl-dATP.

Lane 1: Starting primer

Lane 2: Incorporation of 3-O-azidomethyl-dATPs in presence Mn²±. Lane 3:Extension of the product in lane 2 by addition of all natural dNTPs.Lane 4: Deprotection of the product (0.5 μM) in lane 2 by 300 mM TCEP.Lane 5: Extension of the product in lane 4 by addition of all naturaldNTPs.

g) The Figure depicts a gel showing results of 3′-O-azidomethyl groupdeprotection by 300 mM TCEP after incorporation of3′-O-azidomethyl-dGTP. Lane 1: Starting primer. Lane 2: Incorporation of3-O-azidomethyl-dGTPs in presence Mn²±. Lane 3: Extension of the productin lane 2 by addition of all natural dNTPs. Lane 4: Deprotection of theproduct (0.5 μM) in lane 2 by 300 mM TCEP. Lane 5: Extension of theproduct in lane 4 by addition of all natural dNTPs.

h) Efficiency of deprotection by TCEP on 0.2 μM DNA.

i) Oligonucleotides used for study of the cleavage step.

FIG. 22. Version 2 Chemistry with Double Hairpin Model—Incorporation.

a) Scheme showing incorporation step highlighted in dashed box.

b) Evaluation of DNA polymerases for incorporation of3′-O-modified-dTTPs opposite its natural counterpart. The Figure depictsa gel showing results of incorporation of 3′-O-modified-dTTPs byTherminator IX DNA polymerase at 37° C. Lane 1: Starting material. Lane2: Incorporation of natural dNTP mix. Lane 3: Incorporation of3′-O-azidomethyl-dTTP by Therminator IX DNA polymerase. Lane 4:Extension of the product in lane 3 by addition of all natural dNTPs.

c) Evaluation of DNA polymerases for incorporation of3′-O-modified-dTTPs opposite its natural counterpart. Oligonucleotidesapplicable for study of the incorporation step.

FIG. 23. Version 2 Chemistry with Double Hairpin Model—Cleavage.

a) Scheme showing cleavage of a hairpin Oligonucleotide. Cleavage stepis highlighted in dashed box.

b) Gel showing cleavage of Hairpin Oligonucleotide with Endo V at 37° C.Lane 1. Starting hairpin oligonucleotide. Lane 2 which was the cleavedhairpin oligonucleotide after 5 minutes showed a high yield of digestedDNA with a ratio of ˜98%. Lane 3 which was the cleaved hairpinoligonucleotide after 10 minutes showed a high yield of digested DNAwith a ratio of ˜99%. Lane 4 which was the cleaved hairpinoligonucleotide after 30 minutes showed a high yield of digested DNAwith a ratio of ˜99% and in lane 5 which was the cleaved hairpinoligonucleotide after 1 hr showed a high yield of digested DNA with aratio of ˜99%.

c) Oligonucleotides used for study of the cleavage step.

FIG. 24. Version 2 Chemistry with Double Hairpin Model—Ligation.

a) Scheme showing ligation of hybridized hairpins. Ligation stephighlighted in dashed box.

b) The gel shows ligation of Hairpin Oligonucleotides with Blunt/TA DNAligase at room temperature (24° C.) in the presence of a helper strand.Lane 1 contained a starting hairpin Oligonucleotide. Lane 2 which wasthe ligated hairpin oligonucleotide after 1 minute showed a high yieldof ligated DNA product with a ratio of ˜85%. Lane 3 which was theligated hairpin oligonucleotide after 2 minutes showed a high yield ofdigested DNA with a ratio of ˜85%. Lane 4 which was the ligated hairpinoligonucleotide after 3 minutes showed a high yield of ligated DNAproduct with a ratio of ˜85%. Lane 5 which was the ligated hairpinoligonucleotide after 4 minutes showed a high yield of ligated DNAproduct with a ratio of ˜>85%.

c) Hairpin Oligonucleotides used for study of the Ligation step.

FIG. 25. Version 2 Chemistry—Complete Cycle on Double Hairpin Model.

a) Scheme showing full cycle involving enzymatic incorporation,cleavage, ligation and deprotection steps.

b) Evaluation of DNA polymerases for incorporation of3′-O-modified-dTTPs opposite its natural counterpart. The Figure depictsa gel showing results of incorporation of 3′-O-modified-dTTPs byTherminator IX DNA polymerase at 37° C. Lane 1: Starting material. Lane2: Incorporation of 3′-O-azidomethyl-dTTP by Therminator IX DNApolymerase. Lane 3: Extension of the product in lane 2 by addition ofall natural dNTPs. Lane 4: Cleavage of the product in lane 2 byEndonuclease V. Lane 5: Ligation of the product in lane 4 by blunt TAligase kit.

c) Oligonucleotides applicable for study of the incorporation step.

FIG. 26. Version 2 Chemistry—Complete Cycle on Single Hairpin Modelusing Helper Strand.

a) Scheme showing full cycle involving enzymatic incorporation,cleavage, ligation and deprotection steps.

b) Oligonucleotides applicable for study of the incorporation step.

FIG. 27. Version 3 Chemistry—Complete Cycle on Double-Hairpin Model.

a) Scheme showing full cycle involving enzymatic incorporation,cleavage, ligation and deprotection steps.

b) Oligonucleotides applicable for study of the incorporation step.

FIG. 28. Version 2 Chemistry—Complete Two-Cycle on Double-Hairpin Model.

a) Scheme showing the first full cycle involving enzymaticincorporation, deprotection, cleavage and ligation steps.

b) Scheme showing the second full cycle, following the first full cycle,involving enzymatic incorporation, deprotection, cleavage and ligationsteps.

c) The Figure depicts a gel showing full two-cycle experimentcomprising: incorporation, deprotection, cleavage and ligation steps.

Lane 1. Starting material.Lane 2. Extension of starting material with natural dNTPs.Lane 3. Incorporation of 3′-O-azidomethyl-dTTP by Therminator IX DNApolymerase.Lane 4. Extension of the product in lane 3 by addition of all naturaldNTPs.Lane 5. Deprotection of the product in lane 3 by TCEP.Lane 6. Extension of the product in lane 5 by addition of all naturaldNTPs.Lane 7. Cleavage of the product in lane 5 by Endonuclease V.Lane 8. Ligation of the product in lane 7 by blunt TA ligase kit.Lane 9. Cleavage of the product in lane 8 by Lambda exonuclease.Lane 10. Starting material for second cycle—the same material as in lane9.Lane 11. Incorporation of 3′-O-azidomethyl-dTTP by Therminator IX DNApolymerase.Lane 12. Extension of the product in lane 11 by addition of all naturaldNTPs.Lane 13. Deprotection of the product in lane 11 by TCEP.Lane 14. Extension of the product in lane 13 by addition of all naturaldNTPs.Lane 15. Cleavage of the product in lane 13 by Endonuclease V.Lane 16. Ligation of the product in lane 15 by blunt TA ligase kit.

d) Oligonucleotides used for study.

FIG. 29.

Example showing a mechanism of release from a scaffold polynucleotide ofa polynucleotide of predefined sequence, as synthesised in accordancewith the methods described herein.

FIG. 30.

Schematic of an exemplary method for the synthesis of RNA according tothe invention. The exemplary method shows synthesis in the absence of ahelper strand.

FIG. 31.

Schematic of an exemplary method for the synthesis of RNA according tothe invention. The exemplary method shows synthesis in the presence of ahelper strand.

FIG. 32.

Schematic of an exemplary method for the synthesis of RNA according tothe invention. The exemplary method shows synthesis in the presence of ahelper strand.

FIG. 33.

Schematic of the 1st full cycle of an exemplary method for the synthesisof DNA according to synthesis method version 2 with single hairpinmodel, involving a step of denaturing the helper strand prior to theincorporation step.

FIG. 34.

Schematic of the 2nd full cycle of an exemplary method for the synthesisof DNA according to synthesis method version 2 with single hairpinmodel, involving a step of denaturing the helper strand prior to theincorporation step.

FIG. 35.

Schematic of the 3rd full cycle of an exemplary method for the synthesisof DNA according to synthesis method version 2 with single hairpinmodel, involving a step of denaturing the helper strand prior to theincorporation step.

FIG. 36.

Oligonucleotides used in the experiments detailed in Example 9.

FIG. 37.

Gel showing reaction products corresponding to a full three-cycleexperiment as detailed in Example 9.

The Figure depicts a gel showing the results of a full three-cycleexperiment comprising: incorporation, deblock, cleavage and ligationsteps.

Lane 1: Starting material.Lane 2. Extension of starting material with natural dNTPsLane 3: Incorporation of 3′-O-azidomethyl-dTTP by Therminator X DNApolymerase.Lane 4: Extension of the product in lane 3 by addition of all naturaldNTPs.¬Lane 5: Deblock of the product in lane 3 by TCEPLane 6: Extension of the product in lane 5 by addition of all naturaldNTPs.¬Lane 7: Cleavage of the product in lane 5 by Endonuclease V.Lane 8: Ligation of the product in lane 7 by T3 DNA ligaseLane 9: Starting material for 2nd cycle—the same material as in lane 9Lane 10: Extension of the product in lane 9 by addition of all naturaldNTPs.Lane 11: Incorporation of 3′-O-azidomethyl-dTTP by Therminator X DNApolymerase.Lane 12: Extension of the product in lane 11 by addition of all naturaldNTPs.Lane 13: Deblock of the product in lane 11 by TCEPLane 14: Extension of the product in lane 13 by addition of all naturaldNTPs.Lane 15: Cleavage of the product in lane 13 by Endonuclease VLane 16: Ligation of the product in lane 15 by T3 DNA ligaseLane 17: Starting material for 3rd cycle—the same material as in lane 16Lane 18: Extension of the product in lane 17 by addition of all naturaldNTPs.Lane 19: Incorporation of 3′-O-azidomethyl-dTTP by Therminator X DNApolymerase.Lane 20: Extension of the product in lane 19 by addition of all naturaldNTPs.Lane 21: Deblock of the product in lane 19 by TCEPLane 22: Extension of the product in lane 21 by addition of all naturaldNTPs.Lane 23: Cleavage of the product in lane 21 by Endonuclease VLane 24: Ligation of the product in lane 23 by T3 DNA ligase

FIG. 38.

Fluorescence signals from polyacrylamide gel surfaces spiked withdifferent amount of BRAPA exposed to FITC-PEG-SH and FITC-PEG-COOH.

FIG. 39.

Measured fluorescence signals from fluorescein channel on polyacrylamidegel surfaces spiked with different amount of BRAPA that are exposed toFITC-PEG-SH and FITC-PEG-COOH.

FIG. 40.

(a) Shows sequences of hairpin DNA without linker immobilised ondifferent samples.

(b) Shows sequences of hairpin DNA with linker immobilised on differentsamples.

FIG. 41.

Fluorescence signals from hairpin DNA oligomers with and without linkerimmobilised onto bromoacetyl functionalised polyacrylamide surfaces.

FIG. 42.

Measured fluorescence from hairpin DNA oligomers with and without linkerimmobilised onto bromoacetyl functionalised polyacrylamide surfaces.

FIG. 43.

Fluorescence signals from hairpin DNA oligomers with and without linkerimmobilised onto bromoacetyl functionalised polyacrylamide surfacesfollowing incorporation of triphosphates.

FIG. 44.

Measured fluorescence from hairpin DNA oligomers with and without linkerimmobilised onto bromoacetyl functionalised polyacrylamide surfacesfollowing incorporation of triphosphates.

FIG. 45.

(a) Experimental overview and outcome for each reaction step as detailedin Example 12.

(b) Oligonucleotides used in the experiments detailed in Example 12.

FIG. 46.

Shows fluorescence signals from hairpin DNA oligomers before and aftercleavage reactions (Example 12).

FIG. 47.

Shows measured fluorescence signals from hairpin DNA oligomers beforeand after cleavage reactions (Example 12).

FIG. 48.

Shows the sequences for the inosine-containing strand and thecomplimentary ‘helper’ strand for ligation reactions (Example 12).

FIG. 49.

Results relating to fluorescence signals from hairpin DNA oligomerscorresponding to the monitoring of ligation reactions (Example 12).

FIG. 50.

Results relating to measured fluorescence from hairpin DNA oligomerscorresponding to the monitoring of ligation reactions (Example 12).

FIG. 51.

Results relating to incorporation of 3′-O-modified-dNTPs by TherminatorX DNA polymerase using an incorporation step according to methods of theinvention, e.g. synthesis method versions of the invention 1, 2 and 4(FIGS. 1, 2 and 4 respectively and Example 13).

FIG. 51a provides the nucleic acid sequences of primer strand (primerstrand portion of synthesis strand; SEQ ID NO: 68) and template strand(support strand; SEQ ID NO: 69).

FIG. 51b depicts a gel showing the results of incorporation of3′-O-modified-dNTPs by Therminator X DNA polymerase in presence of Mn2+ions at 37° C.

Lane 1: Starting oligonucleotide.

Lane 2: Incorporation of 3′-O-azidomethyl-dTTP (>99% efficiency) Lane 3:Incorporation of 3′-O-azidomethyl-dATP (>99% efficiency).

Lane 4: Incorporation of 3′-O-azidomethyl-dCTP (>90% efficiency).

Lane 5: Incorporation of 3′-O-azidomethyl-dGTP (>99% efficiency).

Upon addition, the newly added 3′-O-modified-dNTP occupies position n inthe primer strand portion. The next nucleotide position in the primerstrand portion is consequently designated n−1, e.g. in accordance withstep 3 of invention method versions 1, 2 and 4 as depicted in FIGS. 1, 2and 4 respectively.

INTERPRETATION OF FIGURES

The structures depicted in FIGS. 11, 12 a, 13 a, 14 a, 15 a, 16 a, 17 a,18 a, 19 a, 20 a, 21 a, 22 a, 23 a, 24 a, 25 a, 26 a, 27 a, 28 a, 28 b,29, 30, 31, 32, 33, 34, and 35 are to be interpreted consistently withthose depicted in FIGS. 6, 7, 8, 9 and 10. Thus in these Figures, eachleft hand strand of a double-stranded scaffold polynucleotide moleculerelates to the support strand (corresponding to strand “a” in FIGS. 6 to10); each right hand strand of a double-stranded scaffold polynucleotidemolecule relates to the synthesis strand (corresponding to strand “b” inFIGS. 6 to 10); all scaffold polynucleotide molecules comprise a lowersynthesis strand which corresponds to a strand comprising a primerstrand portion (corresponding to the solid and dotted line of strand “b”in FIGS. 6 to 10); certain scaffold polynucleotide molecules (e.g. inFIGS. 15a and 23a ) are shown, prior to incorporation of the newnucleotide, with an upper synthesis strand which corresponds to a strandcomprising a helper strand portion (corresponding to the dashed line ofstrand “b” in FIGS. 6 to 10); certain scaffold polynucleotide molecules(e.g. in FIGS. 12a, 13a and 14a ) are shown with no helper strandportion (corresponding to an absence of the dashed line of strand “b” inFIGS. 6 to 10); and certain scaffold polynucleotide molecules (e.g. inFIGS. 33, 34 and 35) are shown, after the ligation step, with an uppersynthesis strand which corresponds to a strand comprising a helperstrand portion (corresponding to the dashed line of strand “b” in FIGS.6 to 10) and wherein the helper strand portion is removed prior toincorporation of the new nucleotide in the next synthesis cycle.

In addition, in these Figures, where relevant, each new nucleotide isshown to be incorporated together with a reversible terminator group,labelled rtNTP and depicted as a small circular structure (correspondingto the small triangular structure in FIGS. 6 to 10) and terminalphosphate groups are labelled “p” and depicted as a small ellipticalstructure.

FIGS. 11c, 11d, 11g, 11h, 22a, 23a, 24a, 25a, 27a, 28a, 28b , and 29show scaffold polynucleotide molecules wherein strands comprising ahelper strand portion and support strands are connected by a hairpinloop. FIGS. 11b, 22a, 23a, 24a, 25a, 26a, 27a, 28a, 28b , 29, 33, 34,and 35 show scaffold polynucleotide molecules wherein strands comprisinga primer strand portion and support strands are connected by a hairpinloop.

Figures such as FIGS. 27a and 28a show scaffold polynucleotide moleculeswherein the strand comprising a helper strand portion (upper rightstrand) and the support strand (upper left strand) is connected by ahairpin loop and, in the same molecule, the strand comprising the primerstrand portion (lower right strand) and the support strand (lower leftstrand) are connected by a hairpin loop.

The data shown in FIG. 51 are to be interpreted as comprising anincorporation step according to methods of the invention, e.g.consistent with synthesis method versions of the invention 1, 2 and 4(FIGS. 1, 2 and 4 respectively).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for the de novo synthesis ofpolynucleotide molecules according to a predefined nucleotide sequence.Synthesised polynucleotides are preferably DNA and are preferablydouble-stranded polynucleotide molecules. The invention providesadvantages compared with existing synthesis methods. For example, allreaction steps may be performed in aqueous conditions at mild pH,extensive protection and deprotection procedures are not required.Furthermore, synthesis is not dependent upon the copying of apre-existing template strand comprising the predefined nucleotidesequence.

The present inventors have determined that the use of a universalnucleotide, as defined herein, allows a newly-incorporated nucleotide tobe correctly paired with its desired partner nucleotide during eachcycle of synthesis. The use of a universal nucleotide allows for thecreation of a polynucleotide cleavage site within a synthesised regionwhich facilitates cleavage and repeat cycles of synthesis. The inventionprovides versatile methods for synthesising polynucleotides, and forassembling large fragments comprising such synthesised polynucleotides.

Certain embodiments of the synthesis methods of the invention will bedescribed in more general detail herein by reference to exemplarymethods including five exemplary method versions of the invention (FIGS.1 to 5) and certain variants. It is to be understood that all exemplarymethods, including the five exemplary method versions of the invention,are not intended to be limiting on the invention. The invention providesan in vitro method of synthesising a double-stranded polynucleotidemolecule having a predefined sequence, the method comprising performingcycles of synthesis wherein in each cycle a first polynucleotide strandis extended by the incorporation of a nucleotide of the predefinedsequence, and then the second polynucleotide strand which is hybridizedto the first strand is extended by the incorporation of a nucleotidethereby forming a nucleotide pair with the incorporated nucleotide ofthe first strand. Preferably, the methods are for synthesising DNA.Specific methods described herein are provided as embodiments of theinvention.

Reaction Conditions

In one aspect the invention provides a method for synthesising adouble-stranded polynucleotide having a predefined sequence.

In some embodiments, synthesis is carried out under conditions suitablefor hybridization of nucleotides within double-stranded polynucleotides.Polynucleotides are typically contacted with reagents under conditionswhich permit the hybridization of nucleotides to complementarynucleotides. Conditions that permit hybridization are well-known in theart (for example, Sambrook et al., 2001, Molecular Cloning: a laboratorymanual, 3rd edition, Cold Spring Harbour Laboratory Press; and CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York (1995)).

Incorporation of nucleotides into polynucleotides can be carried outunder suitable conditions, for example using a polymerase (e.g.,Therminator IX polymerase) or a terminal deoxynucleotidyl transferase(TdT) enzyme or functional variant thereof to incorporate modifiednucleotides (e.g., 3′-O-modified-dNTPs) at a suitable temperature (e.g.,˜65° C.) in the presence of a suitable buffered solution. In oneembodiment, the buffered solution can comprise 2 mM Tris-HCl, 1 mM(NH₄)₂SO₄, 1 mM KCl, 0.2 mM MgSO₄ and 0.01% Triton® X-100.

Cleavage of polynucleotides can be carried out under suitableconditions, for example using a polynucleotide cleaving enzyme (e.g.,endonuclease) at a temperature that is compatible with the enzyme (e.g.,37° C.) in the presence of a suitable buffered solution. In oneembodiment, the buffered solution can comprise 5 mM potassium acetate, 2mM Tris-acetate, 1 mM magnesium acetate and 0.1 mM DTT.

Ligation of polynucleotides can be carried out under suitableconditions, for example using a ligase (e.g., T4 DNA ligase) at atemperature that is compatible with the enzyme (e.g., room temperature)in the presence of a suitable buffered solution. In one embodiment, thebuffered solution can comprise 4.4 mM Tris-HCl, 7 mM MgCl₂, 0.7 mMdithiothreitol, 0.7 mM ATP, 5% polyethylene glycol (PEG6000).

Deprotection can be carried out under suitable conditions, for exampleusing a reducing agent (e.g., TCEP). For example, deprotection can beperformed using TCEP in Tris buffer (e.g., at a final concentration of300 mM).

Anchor Polynucleotides and Scaffold Polynucleotides

Double-stranded polynucleotides having a predefined sequence aresynthesized by methods of the invention by incorporation of pre-definednucleotides into a pre-existing polynucleotide, referred to herein as ascaffold polynucleotide, which may be attached to or capable of beingattached to a surface as described herein. As described in more detailherein a scaffold polynucleotide forms a support structure toaccommodate the newly-synthesised polynucleotide and, as will beapparent from the description herein, does not comprise a pre-existingtemplate strand which is copied as in conventional methods of synthesis.A scaffold polynucleotide may be referred to as an anchor polynucleotideif the scaffold polynucleotide is attached to a surface. Surfaceattachment chemistries for attaching a scaffold polynucleotide to asurface to form an anchor polynucleotide are described in more detailherein.

In one embodiment a scaffold polynucleotide comprises a synthesis strandhybridized to a complementary support strand. The synthesis strandcomprises a primer strand portion and optionally a helper strand portionseparated by a single-strand break or “nick” (e.g. FIGS. 1 to 5). Boththe primer strand portion and the helper strand portion of the synthesisstrand may be provided hybridized to the complementary support strand.Alternatively, the helper strand portion of the synthesis strand may beprovided separately. The primer strand portion of the synthesis strandmay be provided first, followed by the support strand and helper strand.Alternatively components of the scaffold polynucleotide may be providedseparately. For example, the support strand may be provided first,followed by the primer strand portion of the synthesis strand and thenthe helper strand. The support strand may be provided first, followed bythe helper strand portion of the synthesis strand and then the primerstrand. The helper strand portion may be provided before a cleavagestep. The helper strand portion may be omitted from a scaffoldpolynucleotide prior to cleavage and incorporation of a new predefinednucleotide. The helper strand portion may be removed from a scaffoldpolynucleotide prior to cleavage and incorporation of a new predefinednucleotide, e.g. by denaturation, as describe in more detail herein.Upon mixing of the components in suitable conditions the scaffoldpolynucleotide forms upon hybridization of the separate components.

New synthesis is initiated by polymerase or transferase enzyme at thesite of the single-strand break. Thus polymerase or transferase enzymewill act to extend the terminal nucleotide of the primer strand portionat the site of the single-strand break. The single-stranded break or“nick” between the helper strand portion of the synthesis strand and theprimer strand portion of the synthesis strand is typically initiallyachieved by providing both portions of the synthesis strand as separatemolecules which will align following hybridization with the supportstrand. The (5′) terminal nucleotide of the helper strand at thesingle-stranded break site is typically provided lacking a phosphategroup. The lack of a terminal phosphate group prevents the terminalnucleotide of the helper strand portion ligating with the terminalnucleotide of the primer strand portion at the single-stranded breaksite, thus maintaining the single-stranded break. Creation andmaintenance of the single-stranded break could be effected by othermeans. For example, the terminal nucleotide of the helper strand portionmay be provided with a suitable blocking group which prevents ligationwith the primer strand portion. Preferably the helper strand is providedlacking a terminal phosphate group at the single-stranded break site.

A scaffold polynucleotide may be provided with each of the support andsynthesis strands unconnected at adjacent ends. A scaffoldpolynucleotide may be provided with both support and synthesis strandsconnected at adjacent ends, such as via a hairpin loop, at both ends ofthe scaffold polynucleotide. A scaffold polynucleotide may be providedwith both support and synthesis strands connected at adjacent ends, suchas via a hairpin loop, at one end of the scaffold polynucleotide or anyother suitable linker.

Scaffold polynucleotides with or without hairpins may be immobilized toa solid support or surface as described in more detail herein (see FIG.11).

The terms “hairpin” or “hairpin loop” are commonly used in the currenttechnical field. The term “hairpin loop” is also often referred to as a“stem loop”. Such terms refer to a region of secondary structure in apolynucleotide comprising a loop of unpaired nucleobases which form whenone strand of a polynucleotide molecule hybridizes with another sectionof the same strand due to intramolecular base pairing. Thus hairpins canresemble U-shaped structures. Examples of such structures are shown inFIG. 11.

Nucleotides and Universal Nucleotides

Nucleotides which can be incorporated into synthetic polynucleotides byany of the methods described herein may be nucleotides, nucleotideanalogues and modified nucleotides.

In any of the synthesis methods of the invention defined and describedherein, nucleotides are preferably incorporated as nucleotidescomprising a reversible terminator group as described herein.

Nucleotides may comprise natural nucleobases or non-natural nucleobases.Nucleotides may contain a natural nucleobase, a sugar and a phosphategroup. Natural nucleobases comprise adenosine (A), thymine (T), uracil(U), guanine (G) and cytosine (C). One of the components of thenucleotide may be further modified.

Nucleotide analogues are nucleotides that are modified structurallyeither in the base, sugar or phosphate or combination therein and thatare still acceptable to a polymerase enzyme as a substrate forincorporation into an oligonucleotide strand.

A non-natural nucleobase may be one which will bond, e.g. hydrogen bond,to some degree to all of the nucleobases in the target polynucleotide. Anon-natural nucleobase is preferably one which will bond, e.g. hydrogenbond, to some degree to nucleotides comprising the nucleosides adenosine(A), thymine (T), uracil (U), guanine (G) and cytosine (C).

A non-natural nucleotide may be a peptide nucleic acid (PNA), a lockednucleic acid (LNA) and an unlocked nucleic acid (UNA), a bridged nucleicacid (BNA) or a morpholino, a phosphorothioate or a methylphosphonate.

A non-natural nucleotide may comprise a modified sugar and/or a modifiednucleobase. Modified sugars include but are not limited to2′-O-methylribose sugar. Modified nucleobases include but are notlimited to methylated nucleobases. Methylation of nucleobases is arecognised form of epigenetic modification which has the capability ofaltering the expression of genes and other elements such as microRNAs.Methylation of nucleobases occurs at discrete loci which arepredominately dinucleotide consisting of a CpG motif, but may also occurat CHH motifs (where H is A, C, or T). Typically, during methylation amethyl group is added to the fifth carbon of cytosine bases to createmethylcytosine. Thus modified nucleobases include but are not limited to5-methylcytosine.

Nucleotides of the predefined sequence may be incorporated oppositepartner nucleotides to form a nucleotide pair. A partner nucleotide maybe a complementary nucleotide. A complementary nucleotide is anucleotide which is capable of bonding, e.g. hydrogen bonding, to somedegree to the nucleotides of the predefined sequence.

Typically, a nucleotide of the predefined sequence is incorporated intoa polynucleotide opposite a naturally complementary partner nucleobase.Thus adenosine may be incorporated opposite thymine and vice versa.Guanine may be incorporated opposite cytosine and vice versa.Alternatively, a nucleotide of the predefined sequence may beincorporated opposite a partner nucleobase to which it will bond, e.g.hydrogen bond, to some degree.

Alternatively a partner nucleotide may be a non-complementarynucleotide. A non-complementary nucleotide is a nucleotide which is notcapable of bonding, e.g. hydrogen bonding, to the nucleotide of thepredefined sequence. Thus a nucleotide of the predefined sequence may beincorporated opposite a partner nucleotide to form a mismatch, providedthat the synthesised polynucleotide overall is double-stranded andwherein the first strand is attached to the second strand byhybridization.

The term “opposite” is to be understood as relating to the normal use ofthe term in the field of nucleic acid biochemistry, and specifically toconventional Watson-Crick base-pairing. Thus a first nucleic acidmolecule of sequence 5′-ACGA-3′ may form a duplex with a second nucleicacid molecule of sequence 5′-TCGT-3′ wherein the G of the first moleculewill be positioned opposite the C of the second molecule and willhydrogen bond therewith. A first nucleic acid molecule of sequence5′-ATGA-3′ may form a duplex with a second nucleic acid molecule ofsequence 5′-TCGT-3′, wherein the T of the first molecule will mismatchwith the G of the second molecule but will still be positioned oppositetherewith and will act as a partner nucleotide. This principle appliesto any nucleotide partner pair relationship disclosed herein, includingpartner pairs comprising universal nucleotides.

In all of the methods described herein a position in the support strand,and the opposite position in the synthesis strand, is assigned theposition number “n”. This position refers to the position of anucleotide in the support strand of a scaffold polynucleotide which inany given synthesis cycle is opposite the nucleotide position in thesynthesis strand which is occupied by or will be occupied by thefirst/next nucleotide of the predefined sequence upon its addition tothe terminal end of the primer strand portion in that cycle at step (3)or in incorporation steps of subsequent cycles. Position “n” also refersto the position in the support strand of a ligation polynucleotide atligation step (5), or in ligation steps of subsequent cycles, whichposition is the nucleotide position which will be opposite thefirst/subsequent nucleotide of the predefined sequence upon ligation ofthe ligation polynucleotide to the cleaved scaffold polynucleotide instep (5) of the first cycle or in ligation steps of subsequent cycles.

Both the position in the support strand and the opposite position in thesynthesis strand may be referred to as positon n.

Further details concerning the definition of position “n” are providedwith reference to FIGS. 1 to 5 and the descriptions thereof in relationto the five exemplary synthesis method versions 1 to 5 described in moredetail herein.

Nucleotides and nucleotide analogues may preferably be provided asnucleoside triphosphates. Thus in any of the methods of the invention inorder to synthesise DNA polynucleotides, nucleotides may be incorporatedfrom 2′-deoxyribonucleoside-5′-O-triphosphates (dNTPs), e.g. via theaction of a DNA polymerase enzyme or e.g. via the action of an enzymehaving deoxynucleotidyl terminal transferase activity. In any of themethods of the invention in order to synthesise RNA polynucleotides,nucleotides may be incorporated ribonucleoside-5′-O-triphosphates(NTPs), e.g. via the action of a RNA polymerase enzyme or e.g. via theaction of an enzyme having nucleotidyl terminal transferase activity.Triphosphates can be substituted by tetraphosphates or pentaphosphates(generally oligophosphate). These oligophosphates can be substituted byother alkyl or acyl groups:

Methods of the invention may use a universal nucleotide. A universalnucleotide may be used as a component of the support strand of ascaffold molecule to facilitate a newly-incorporated nucleotide to becorrectly paired with its desired partner nucleotide during each cycleof synthesis. A universal nucleotide may also be incorporated into thesynthesis strand as a component of the predefined nucleotide sequence ifdesired.

A universal nucleotide is one wherein the nucleobase will bond, e.g.hydrogen bond, to some degree to the nucleobase of any nucleotide of thepredefined sequence. A universal nucleotide is preferably one which willbond, e.g. hydrogen bond, to some degree to nucleotides comprising thenucleosides adenosine (A), thymine (T), uracil (U), guanine (G) andcytosine (C). The universal nucleotide may bond more strongly to somenucleotides than to others. For instance, a universal nucleotide (I)comprising the nucleoside, 2′-deoxyinosine, will show a preferentialorder of pairing of I-C>I-A>I-G approximately =I-T.

Examples of possible universal nucleotides are inosines ornitro-indoles. The universal nucleotide preferably comprises one of thefollowing nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole,6-nitroindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole,4-nitrobenzimidazole, 5-nitroindazole, 4-aminobenzimidazole or phenyl(C6-aromatic ring. The universal nucleotide more preferably comprisesone of the following nucleosides: 2′-deoxyinosine, inosine,7-deaza-2′-deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine,2-aza-inosine, 4-nitroindole 2′-deoxyribonucleoside, 4-nitroindoleribonucleoside, 5-nitroindole 2′ deoxyribonucleoside, 5-nitroindoleribonucleoside, 6-nitroindole 2′ deoxyribonucleoside, 6-nitroindoleribonucleoside, 3-nitropyrrole 2′ deoxyribonucleoside, 3-nitropyrroleribonucleoside, an acyclic sugar analogue of hypoxanthine,nitroimidazole 2′ deoxyribonucleoside, nitroimidazole ribonucleoside,4-nitropyrazole 2′ deoxyribonucleoside, 4-nitropyrazole ribonucleoside,4-nitrobenzimidazole 2′ deoxyribonucleoside, 4-nitrobenzimidazoleribonucleoside, 5-nitroindazole 2′ deoxyribonucleoside, 5-nitroindazoleribonucleoside, 4-aminobenzimidazole 2′ deoxyribonucleoside,4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside or phenylC-2′-deoxyribosyl nucleoside.

Some examples of universal bases are shown below:

Universal nucleotides incorporating cleavable bases may also be used,including photo- and enzymatically-cleavable bases, some examples ofwhich are shown below.

Photocleavable Bases:

Base Analogues Cleavable by Endonuclease III:

Base Analogues Cleavable by Formamidopyrimidine DNA Glycosylase (Fpg):

Base Analogues Cleavable by 8-Oxoguanine DNA Glycosylase (hOGG1):

Base Analogues Cleavable by hNeil1:

Base Analogues Cleavable by Thymine DNA Glycosylase (TDG):

Base Analogues Cleavable by Human Alkyladenine DNA Glycosylase (hAAG):

Bases Cleavable by Uracil DNA Glycosylase:

Bases Cleavable by Human Single-Strand-Selective MonofunctionalUracil-DNA Glycosylase (SMUG1):

Bases Cleavable by 5-methylcytosine DNA Glycosylase (ROS1):

(see S. S. David, S. D. Williams Chemical reviews 1998, 98, 1221-1262and M. I. Ponferrada-Marín, T. Roldán-Arjona, R. R. Ariza'Nucleic AcidsRes 2009, 37, 4264-4274).

In any of the methods involving scaffold polynucleotides, the universalnucleotide most preferably comprises 2′-deoxyinosine.

Examples of epigenetic bases which may be incorporated using any of thesynthesis methods described herein include the following:

Examples of modified bases which may be incorporated using any of thesynthesis methods described herein include the following:

Examples of halogenated bases which may be incorporated using any of thesynthesis methods described herein include the following:

where R1=F, Cl, Br, I, alkyl, aryl, fluorescent label, aminopropargyl,aminoallyl.

Examples of amino-modified bases, which may be useful in e.g.attachment/linker chemistry, which may be incorporated using any of thesynthesis methods described herein include the following:

where base=A, T, G or C with alkyne or alkene linker.

Examples of modified bases, which may be useful in e.g. click chemistry,which may be incorporated using any of the synthesis methods describedherein include the following:

Examples of biotin-modified bases which may be incorporated using any ofthe synthesis methods described herein include the following:

where base=A, T, G or C with alkyne or alkene linker.

Examples of bases bearing fluorophores and quenchers which may beincorporated using any of the synthesis methods described herein includethe following:

Nucleotide-Incorporating Enzymes

Enzymes are available that are capable of extending, by the addition ofa nucleotide, a single-stranded polynucleotide portion of adouble-stranded polynucleotide molecule and/or that are capable ofextending one strand of a blunt-ended double-stranded polynucleotidemolecule. This includes enzymes which have template-independent enzymeactivity, such as template-independent polymerase ortemplate-independent transferase activity.

Thus in any of the methods described herein the enzyme which is used forthe addition of a nucleotide of the predefined sequence, e.g. to theterminal end of the synthesis strand of a scaffold polynucleotide, hastemplate-independent enzyme activity, such as template-independentpolymerase or template-independent transferase activity.

Any suitable enzyme may be employed to add a predefined nucleotide usingthe methods described herein. Thus in all methods defined and describedherein referring to the use of a polymerase or a transferase enzyme, thepolymerase or transferase enzyme may be substituted with another enzymecapable of performing the same function as a polymerase or transferaseenzyme in the context of the methods of the invention.

A polymerase enzyme may be employed in the methods described herein.Polymerase enzymes may be chosen based on their ability to incorporatemodified nucleotides, in particular nucleotides having attachedreversible terminator groups, as described herein. In the exemplarymethods described herein all polymerases which act on DNA must not have3′ to 5′ exonuclease activity. The polymerase may have stranddisplacement activity.

Thus preferably the polymerase is a modified polymerase having anenhanced ability to incorporate a nucleotide comprising a reversibleterminator group compared to an unmodified polymerase. The polymerase ismore preferably a genetically engineered variant of the native DNApolymerase from Thermococcus species 9° N, preferably species 9° N-7.Examples of modified polymerases are Therminator IX DNA polymerase andTherminator X DNA polymerase available from New England BioLabs. Thisenzyme has an enhanced ability to incorporate 3′-O-modified dNTPs.

Examples of other polymerases that can be used for incorporation ofreversible terminator dNTPs in any of the methods of the invention areDeep Vent (exo-), Vent (Exo-), 9° N DNA polymerase, Therminator DNApolymerase, Therminator IX DNA polymerase, Therminator X DNA polymerase,Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNA polymerase,Sulfolobus DNA polymerase I, and Taq Polymerase.

Examples of other polymerases that can be used for incorporation ofreversible terminator NTPs in any of the methods of the invention are T3RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, pol lambda, polmicro or 129 DNA polymerase.

For the extension of such a polynucleotide synthesis molecule comprisingDNA, a DNA polymerase may be used. Any suitable DNA polymerase may beused.

The DNA polymerase may be for example Bst DNA polymerase full length,Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment, E.coli DNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reversetranscriptase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, TaqDNA polymerase, T4 DNA polymerase, T7 DNA polymerase and enzymes havingreverse transcriptase activity, for example M-MuLV reversetranscriptase.

The DNA polymerase may lack 3′ to 5′ exonuclease activity. Any suchsuitable polymerase enzyme may be used. Such a DNA polymerase may be,for example, Bst DNA polymerase full length, Bst DNA polymerase largefragment, Bsu DNA polymerase large fragment, DNA Pol I large (Klenow)fragment (3′→5′ exo-), M-MuLV reverse transcriptase, Sulfolobus DNApolymerase IV, Taq DNA polymerase.

The DNA polymerase may possess strand displacement activity. Any suchsuitable polymerase enzyme may be used. Such a DNA polymerase may be,for example, Bst DNA polymerase large fragment, Bsu DNA polymerase largefragment, DNA Pol I large (Klenow) fragment (3′→5′ exo-), M-MuLV reversetranscriptase, phi29 DNA polymerase.

The DNA polymerase may lack 3′ to 5′ exonuclease activity and maypossess strand displacement activity. Any such suitable polymeraseenzyme may be used. Such a DNA polymerase may be, for example, Bst DNApolymerase large fragment, Bsu DNA polymerase large fragment, E. coliDNA polymerase DNA Pol I large (Klenow) fragment, M-MuLV reversetranscriptase.

The DNA polymerase may lack 5′ to 3′ exonuclease activity. Any suchsuitable polymerase enzyme may be used. Such a DNA polymerase may be,for example, Bst DNA polymerase large fragment, Bsu DNA polymerase largefragment, DNA Pol I large (Klenow) fragment, DNA Pol I large (Klenow)fragment (3′→5′ exo-), M-MuLV reverse transcriptase, phi29 DNApolymerase, Sulfolobus DNA polymerase IV, T4 DNA polymerase, T7 DNApolymerase.

The DNA polymerase may lack both 3′ to 5′ and 5′ to 3′ exonucleaseactivities and may possess strand displacement activity. Any suchsuitable polymerase enzyme may be used. Such a DNA polymerase may be,for example, Bst DNA polymerase large fragment, Bsu DNA polymerase largefragment, DNA Pol I large (Klenow) fragment (3′→5′ exo-), M-MuLV reversetranscriptase.

The DNA polymerase may also be a genetically engineered variant. Forexample, the DNA polymerase may be a genetically engineered variant. ofthe native DNA polymerase from Thermococcus species 9° N, such asspecies 9° N-7. One such example of a modified polymerase is TherminatorIX DNA polymerase or Therminator X DNA polymerase available from NewEngland BioLabs. Other engineered or variant DNA polymerases includeDeep Vent (exo-), Vent (Exo-), 9° N DNA polymerase, Therminator DNApolymerase, Klenow fragment (Exo-), Bst DNA polymerase, Bsu DNApolymerase, Sulfolobus DNA polymerase I, and Taq Polymerase.

For the extension of such a polynucleotide synthesis molecule comprisingRNA, any suitable enzyme may be used. For example an RNA polymerase maybe used. Any suitable RNA polymerase may be used.

The RNA polymerase may be T3 RNA polymerase, T7 RNA polymerase, SP6 RNApolymerase, E. coli RNA polymerase holoenzyme.

The enzyme may have a terminal transferase activity, e.g. the enzyme maybe a terminal nucleotidyl transferase, or terminal deoxynucleotidyltransferase, and wherein the polynucleotide synthesis molecule isextended to form a polynucleotide molecule comprising DNA or RNA,preferably DNA. Any of these enzymes may be used in the methods of theinvention wherein extension of a polynucleotide synthesis molecule isrequired.

One such enzyme is a terminal nucleotidyl transferase enzyme, such asterminal deoxynucleotidyl transferase (TdT) (see e.g. Motea et al, 2010;Minhaz Ud-Dean, Syst. Synth. Biol., 2008, 2(3-4), 67-73). TdT is capableof catalysing the addition to a polynucleotide synthesis molecule of anucleotide molecule (nucleoside monophosphate) from a nucleosidetriphosphate substrate (NTP or dNTP). TdT is capable of catalysing theaddition of natural and non-natural nucleotides. It is also capable ofcatalysing the addition of nucleotide analogues (Motea et al, 2010). Pollambda and pol micro enzymes may also be used (Ramadan K, et al., J.Mol. Biol., 2004, 339(2), 395-404), as may 129 DNA polymerase.

Techniques for the extension of a single-stranded polynucleotidemolecule, both DNA and RNA, in the absence of a template by the actionof a terminal transferase enzyme (e.g. terminal deoxynucleotidyltransferase; TdT) to create an artificially-synthesised single-strandedpolynucleotide molecule has been extensively discussed in the art. Suchtechniques are disclosed in, for example, Patent applicationpublications WO2016/034807, WO 2016/128731, WO2016/139477 andWO2017/009663, as well as US2014/0363852, US2016/0046973,US2016/0108382, and US2016/0168611. These documents describe thecontrolled extension of a single-stranded polynucleotide synthesismolecule by the action of TdT to create an artificially-synthesisedsingle-stranded polynucleotide molecule. Extension by natural andnon-natural/artificial nucleotides using such enzymes is described, asis extension by modified nucleotides, for example, nucleotidesincorporating blocking groups. Any of the terminal transferase enzymesdisclosed in these documents may be applied to methods of the presentinvention, as well as any enzyme fragment, derivative, analogue orfunctional equivalent thereof provided that the terminal transferasefunction is preserved in the enzyme.

Directed evolution techniques, conventional screening, rational orsemi-rational engineering/mutagenesis methods or any other suitablemethods may be used to alter any such enzyme to provide and/or optimisethe required function. Any other enzyme which is capable of extending asingle-stranded polynucleotide molecule portion, such as a moleculecomprising DNA or RNA, with a nucleotide without the use of a templatemay be used.

Thus in any of the methods defined herein a single strandedpolynucleotide synthesis molecule portion comprising DNA or blunt-endeddouble-stranded polynucleotide comprising DNA may be extended by anenzyme which has template-independent enzyme activity, such astemplate-independent polymerase or transferase activity. The enzyme mayhave nucleotidyl transferase enzyme activity, e.g. a deoxynucleotidyltransferase enzyme, such as terminal deoxynucleotidyl transferase (TdT),or an enzyme fragment, derivative, analogue or functional equivalentthereof. A polynucleotide synthesis molecule extended by the action ofsuch an enzyme comprises DNA.

In any of the methods defined herein a single stranded portion of apolynucleotide synthesis molecule comprising RNA, or blunt-endeddouble-stranded polynucleotide comprising RNA may be extended by anenzyme which has nucleotidyl transferase enzyme (e.g including TdT), oran enzyme fragment, derivative, analogue or functional equivalentthereof. A polynucleotide synthesis molecule extended by the action ofsuch an enzyme may comprise RNA. For the synthesis of a single strandedpolynucleotide synthesis molecule comprising RNA, or a single strandedportion of a polynucleotide synthesis molecule comprising RNA, anysuitable nucleotidyl transferase enzyme may be used. Nucleotidyltransferase enzymes such as poly (U) polymerase and poly(A) polymerase(e.g. from E. coli) are capable of template-independent addition ofnucleoside monophosphate units to polynucleotide synthesis molecules.Any of these enzymes may be applied to methods of the present invention,as well as any enzyme fragment, derivative, analogue or functionalequivalent thereof provided that the nucleotidyl transferase function ispreserved in the enzyme. Directed evolution techniques, conventionalscreening, rational or semi-rational engineering/mutagenesis methods orany other suitable methods may be used to alter any such enzyme toprovide and/or optimise the required function.

Reversible Blocking Groups

All methods defined and described herein refer to a reversible blockinggroup or reversible terminator group. Such groups act to prevent furtherextension by the enzyme in a given synthesis cycle so that only anucleotide of predefined sequence may controllably be used to extend thesynthesis strand, and thus non-specific nucleotide incorporation isprevented. Any functionality which achieves this effect may be used inany of the methods defined and described herein. Reversible blockinggroups/reversible terminator groups attached to nucleotides anddeblocking steps are preferred means for achieving this effect. Howeverthis effect may be achieved by alternative means as appropriate.

Any suitable reversible blocking group may be attached to a nucleotideto prevent further extension by the enzyme following the incorporationof a nucleotide in a given cycle and to limit incorporation to onenucleotide per cycle. In any the methods of the invention the reversibleblocking group is preferably a reversible terminator group which acts toprevent further extension by a polymerase enzyme. Examples of reversibleterminators are provided below.

Propargyl Reversible Terminators:

Allyl Reversible Terminators:

Cyclooctene Reversible Terminators:

Cyanoethyl Reversible Terminators:

Nitrobenzyl Reversible Terminators:

Disulfide Reversible Terminators:

Azidomethyl Reversible Terminators:

Aminoalkoxy Reversible Terminators:

Nucleoside triphosphates with bulky groups attached to the base canserve as substitutes for a reversible terminator group on 3′-hydroxygroup and can block further incorporation. This group can be deprotectedby TCEP or DTT producing natural nucleotides.

For synthesising DNA polynucleotides according to any of the methods ofthe invention preferred modified nucleosides are3′-O-modified-2′-deoxyribonucleoside-5′-O-triphosphate. For synthesisingRNA polynucleotides according to any of the methods of the inventionpreferred modified nucleosides are3′-O-modified-ribonucleoside-5′-O-triphosphate.

Preferred modified dNTPs are modified dNTPs which are 3′-O-allyl-dNTPsand 3′-O-azidomethyl-dNTPs.

3′-O-allyl-dNTPs are shown below.

3′-O-azidomethyl-dNTPs are shown below.

Methods of the invention described and defined herein may refer to adeprotection or deblocking step. Such a step involves removal of thereversible blocking group (e.g. the reversible terminator group) by anysuitable means, or otherwise reversing the functionality of theblocking/terminator group to inhibit further extension by theenzyme/polymerase.

Any suitable reagent may be used to remove the reversible terminatorgroup at the deprotection step.

A preferred deprotecting reagent is tris(carboxyethyl)phosphine (TCEP).TCEP may be used to remove reversible terminator groups from3′-O-allyl-nucleotides (in conjunction with Pd⁰) and3′-O-azidomethyl-nucleotides following incorporation.

Examples of deprotecting reagents are provided below.

Propargyl Reversible Terminators:

Treatment by Pd catalysts—Na₂PdCl₄, PdCl₂Ligands can be used e. g.: Triphenylphosphine-3,3′,3″-trisulfonic acidtrisodium salt.

Allyl Reversible Terminators:

Treatment by Pd catalysts—Na₂PdCl₄, PdCl₂Ligands can be used e. g.: Triphenylphosphine-3,3′,3″-trisulfonic acidtrisodium salt.

Azidomethyl Reversible Terminators:

Treatment by thiol (mercaptoethanol or dithiothreitol), or Tris(2-carboxyethyl)phosphine-TCEP.

Cyanoethyl Reversible Terminators:

Treatment by fluoride-ammonium fluoride, tetrabutylammonium fluoride(TBAF).

Nitrobenzyl Reversible Terminators:

Exposure to UV light

Disulfide Reversible Terminators:

Treatment by thiol (mercaptoethanol or dithiothreitol), or Tris(2-carboxyethyl)phosphine-TCEP.

Aminoalkoxy Reversible Terminators:

Treatment by nitrite (NO₂ ⁻, HNO₂) pH=5.5

A reversible blocking group (e.g., a reversible terminator group) can beremoved by a step performed immediately after the incorporation step andbefore the cleavage step, provided that unwanted reagent from theincorporation step is removed to prevent further incorporation followingremoval of the reversible terminator group. A reversible blocking group(e.g., a reversible terminator group) can be removed by a step performedimmediately after the cleavage step and before the ligation step. Areversible blocking group (e.g., a reversible terminator group) can beremoved by a step performed immediately after the ligation step.

Synthetic Polynucleotide

The polynucleotide having a predefined sequence synthesised according tothe methods described herein is double-stranded. The synthesisedpolynucleotide overall is double-stranded and wherein the first strandis attached to the second strand by hybridization. Mismatches andregions of non-hybridization may be tolerated, provided that overall thefirst strand is attached to the second strand by hybridization.

Hybridisation may be defined by moderately stringent or stringenthybridisation conditions. A moderately stringent hybridisation conditionuses a prewashing solution containing 5× sodium chloride/sodium citrate(SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridisation buffer of about 50%formamide, 6×SSC, and a hybridisation temperature of 55° C. (or othersimilar hybridisation solutions, such as one containing about 50%formamide, with a hybridisation temperature of 42° C.), and washingconditions of 60° C., in 0.5×SSC, 0.1% SDS. A stringent hybridisationcondition hybridises in 6×SSC at 45° C., followed by one or more washesin 0.1×SSC, 0.2% SDS at 68° C.

The double-stranded polynucleotide having a predefined sequencesynthesised according to the methods described herein may be retained asa double-stranded polynucleotide. Alternatively the two strands of thedouble-stranded polynucleotide may be separated to provide asingle-stranded polynucleotide having a predefined sequence. Conditionsthat permit separation of two strands of a double-strandedpolynucleotide (melting) are well-known in the art (for example,Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rdedition, Cold Spring Harbour Laboratory Press; and Current Protocols inMolecular Biology, Greene Publishing and Wiley-lnterscience, New York(1995)).

The double-stranded polynucleotide having a predefined sequencesynthesised according to the methods described herein may be amplifiedfollowing synthesis. Any region of the double-stranded polynucleotidemay be amplified. The whole or any region of the double-strandedpolynucleotide may be amplified together with the whole or any region ofthe scaffold polynucleotide. Conditions that permit amplification of adouble-stranded polynucleotide are well-known in the art (for example,Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rdedition, Cold Spring Harbour Laboratory Press; and Current Protocols inMolecular Biology, Greene Publishing and Wiley-Interscience, New York(1995)). Thus any of the synthesis methods described herein may furthercomprise an amplification step wherein the double-strandedpolynucleotide having a predefined sequence, or any region thereof, isamplified as described above. Amplification may be performed by anysuitable method, such as polymerase chain reaction (PCR), polymerasespiral reaction (PSR), loop mediated isothermal amplification (LAMP),nucleic acid sequence based amplification (NASBA), self-sustainedsequence replication (3SR), rolling circle amplification (RCA), stranddisplacement amplification (SDA), multiple displacement amplification(MDA), ligase chain reaction (LCR), helicase dependant amplification(HDA), ramification amplification method (RAM) etc. Preferably,amplification is performed by polymerase chain reaction (PCR).

The double-stranded or single-stranded polynucleotide having apredefined sequence synthesised according to the methods describedherein can be any length. For example, the polynucleotides can be atleast 10, at least 50, at least 100, at least 150, at least 200, atleast 250, at least 300, at least 350, at least 400, at least 450 or atleast 500 nucleotides or nucleotide pairs in length. For example, thepolynucleotides may be from about 10 to about 100 nucleotides ornucleotide pairs, about 10 to about 200 nucleotides or nucleotide pairs,about 10 to about 300 nucleotides or nucleotide pairs, about 10 to about400 nucleotides or nucleotide pairs and about 10 to about 500nucleotides or nucleotide pairs in length. The polynucleotides can be upto about 1000 or more nucleotides or nucleotide pairs, up to about 5000or more nucleotides or nucleotide pairs in length or up to about 100000or more nucleotides or nucleotide pairs in length.

Cleavage of Scaffold Polynucleotide

In methods requiring the presence of scaffold polynucleotides and stepsof cleavage prior to ligation, the selection of the reagent to performthe cleavage step will depend upon the particular method employed. Thecleavage site is defined by the specific position of the universalnucleotide in the support strand and the requirement for a blunt-endedscaffold polynucleotide once cleaved or the requirement for an overhangin the scaffold polynucleotide once cleaved. Configuration of thedesired cleavage site and selection of the appropriate cleavage reagentwill therefore depend upon the specific chemistry employed in themethod, as will readily be apparent by reference to the exemplarymethods described herein.

Some examples of DNA cleaving enzymes recognizing modified bases areshown in the Table below.

DNA Termini created from glycosylase/ Main the cleavage Endonucleasesubstrate Cleavage site 5′-end 3′-end APE1 AP site 1st phospho-Deoxyribose- OH diester bond 5' 5′-phosphate to the lesion Endo- APsite, 1st phospho- phosphate 3′-phospho-α, nuclease III thymine diesterbond 3′ β-unsaturated glycol to the lesion aldehyde Endo- AP site 1stphospho- Deoxyribose- OH nuclease IV diester bond 5′ 5′-phosphate to thelesion Endo- Inosine 2nd phospho- phosphate OH nuclease V diester bond3′ to the lesion Endo- AP site, 1st phospho- phosphate phosphatenuclease thymine diester bond VIII glycol 5′ and 3′ to the lesion FpG8-oxo- 1st phospho- phosphate phosphate guanine diester bond 5′ and 3′to the lesion hOGG1 8-oxo- 1st phospho- phosphate 3′-phospho-α, guaninediester bond 3′ β-unsaturated to the lesion aldehyde hNeil1 Oxidized 1stphospho- phosphate phosphate purines diester bond 5′ and 3′ to thelesion ROS1 5- 1st phospho- phosphate phosphate methyl- diester bondcytosine 5′ and 3′ to the lesion Uracil DNA Uracil N-glycosidic AP site(no break) glycosylase bond hSMUG Uracil N-glycosidic AP site (no break)bond hAAG Inosine N-glycosidic AP site (no break) bond

Ligation Polynucleotide

In methods requiring the presence of scaffold polynucleotides and stepsof ligation following cleavage, the selection of the configuration andstructure of the ligation polynucleotide will also depend upon theparticular method employed. The ligation polynucleotide generallycomprises a support strand as described herein and a helper strand asdescribed herein. The support strand and the helper strand used in theligation polynucleotide can be the same or different from those used inthe initial scaffold polynucleotide construct. For example, therequirement for a single- or double-nucleotide overhang in the supportstrand of the ligation end of the ligation polynucleotide will dependupon the method employed. The appropriate structure can readily beachieved by reference to the exemplary methods described herein.

The complementary ligation end of the ligation polynucleotide istypically provided with a non-phosphorylated terminal nucleotide in thehelper strand adjacent the overhang. This prevents ligation of thehelper strand portion of the synthesis strand to the primer strandportion of the synthesis strand and thus maintains the single-strandbreak in the synthesis strand. Alternative means for preventing ligationin the synthesis strands could be employed. For example blockingmoieties could be attached to the terminal nucleotide in the helperstrand. Moreover, the helper stand may be removed from the scaffoldmolecule, e.g. by denaturation, prior to cleavage and incorporation ofthe next predefined nucleotide in the next synthesis cycle, as describedfurther herein.

Ligation

In methods of the invention which involve a ligation step, ligation maybe achieved using any suitable means. Preferably, the ligation step willbe performed by a ligase enzyme. The ligase may be a modified ligasewith enhanced activity for single-base overhang substrates. The ligasemay be a T3 DNA ligase or a T4 DNA ligase. The ligase may a blunt TAligase. For example a blunt TA ligase is available from New EnglandBioLabs (NEB). This is a ready-to-use master mix solution of T4 DNALigase, ligation enhancer, and optimized reaction buffer specificallyformulated to improve ligation and transformation of both blunt-end andsingle-base overhang substrates. Molecules, enzymes, chemicals andmethods for ligating (joining) single- and double-strandedpolynucleotides are well known to the skilled person.

Solid Phase Synthesis

Synthetic polynucleotides produced in accordance with the synthesismethods of the invention may preferably be synthesised using solid phaseor reversible solid phase techniques. A variety of such techniques isknown in the art and may be used. Before initiating synthesis of a newdouble-stranded polynucleotide of predefined sequence, scaffoldpolynucleotides may be immobilized to a surface e.g. a planar surfacesuch as glass, a gel-based material, or the surface of a microparticlesuch as a bead or functionalised quantum dot. The material comprisingthe surface may itself be bound to a substrate. For example, scaffoldpolynucleotides may be immobilized to a gel-based material such as e.g.polyacrylamide, and wherein the a gel-based material is bound to asupporting substrate such as glass.

Polynucleotides may be immobilized or tethered to surfaces directly orindirectly. For example they may be attached directly to surfaces bychemical bonding. They may be indirectly tethered to surfaces via anintermediate surface, such as the surface of a microparticle or beade.g. as in SPRI or as in electrowetting systems, as described below.Cycles of synthesis may then be initiated and completed whilst thescaffold polynucleotide incorporating the newly-synthesisedpolynucleotide is immobilized.

In such methods a double-stranded scaffold polynucleotide may beimmobilized to a surface prior to the incorporation of the firstnucleotide of the predefined sequence. Such an immobilizeddouble-stranded scaffold polynucleotide may therefore act as an anchorto tether the double-stranded polynucleotide of the predefined sequenceto the surface during and after synthesis.

Only one strand of such a double-stranded anchor/scaffold polynucleotidemay be immobilized to the surface at the same end of the molecule.Alternatively both strands of a double-stranded anchor/scaffoldpolynucleotide may each be immobilized to the surface at the same end ofthe molecule. A double-stranded anchor/scaffold polynucleotide may beprovided with each strand connected at adjacent ends, such as via ahairpin loop at the opposite end to the site of initiation of newsynthesis, and connected ends may be immobilized on a surface (forexample as depicted schematically in FIG. 11).

In methods involving a scaffold polynucleotide, as described herein, thescaffold polynucleotide may be attached to a surface prior to theincorporation of the first nucleotide in the predefined sequence. Thusthe synthesis strand comprising the primer strand portion and theportion of the support strand hybridized thereto may both be separatelyattached to a surface, as depicted in FIGS. 11(a) and (c). The synthesisstrand comprising the primer strand portion and the portion of thesupport strand hybridized thereto may be connected at adjacent ends,such as via a hairpin loop, e.g. at the opposite end to the site ofinitiation of new synthesis, and connected ends may be tethered to asurface, as depicted in FIGS. 11(b) and (d). One or other of thesynthesis strand comprising the primer strand portion and the portion ofthe support strand hybridized thereto may be attached separately to asurface, as depicted in FIG. 11(e) to (h). Preferably the synthesisstrand comprising the primer strand portion and the portion of thesupport strand hybridized thereto is attached to a surface.

Solid Phase Synthesis on Planar Surfaces

Before initiating synthesis of a new double-stranded polynucleotide ofpredefined sequence synthetic anchor/scaffold polynucleotides can besynthesised by methods known in the art, including those describedherein, and tethered to a surface.

Pre-formed polynucleotides can be tethered to surfaces by methodscommonly employed to create nucleic acid microarrays attached to planarsurfaces. For example, anchor/scaffold polynucleotides may be createdand then spotted or printed onto a planar surface. Anchor/scaffoldpolynucleotides may be deposited onto surfaces using contact printingtechniques. For example, solid or hollow tips or pins may be dipped intosolutions comprising pre-formed scaffold polynucleotides and contactedwith the planar surface. Alternatively, oligonucleotides may be adsorbedonto micro-stamps and then transferred to a planar surface by physicalcontact. Non-contact printing techniques include thermic printing orpiezoelectric printing wherein sub-nanolitre size microdropletscomprising pre-formed scaffold polynucleotides may be ejected from aprinting tip using methods similar to those used in inkjet and bubblejetprinting.

Single-stranded oligonucleotides may be synthesised directly on planarsurfaces such as using so-called “on-chip” methods employed to createmicroarrays. Such single-stranded oligonucleotides may then act asattachment sites to immobilize pre-formed anchor/scaffoldpolynucleotides.

On-chip techniques for generating single-stranded oligonucleotidesinclude photolithography which involves the use of UV light directedthrough a photolithographic mask to selectively activate a protectednucleotide allowing for the subsequent incorporation of a new protectednucleotide. Cycles of UV-mediated deprotection and coupling ofpre-determined nucleotides allows the in situ generation of anoligonucleotide having a desired sequence. As an alternative to the useof a photolithographic mask, oligonucleotides may be created on planarsurfaces by the sequential deposition of nucleobases using inkjetprinting technology and the use of cycles of coupling, oxidation anddeprotection to generate an oligonucleotide having a desired sequence(for a review see Kosuri and Church, Nature Methods, 2014, 11, 499-507).

In any of the synthesis methods described herein, including methodsinvolving reversible immobilisation as described below, surfaces can bemade of any suitable material. Typically a surface may comprise silicon,glass or polymeric material. A surface may comprise a gel surface, suchas a polyacrylamide surface, such as about 2% polyacrylamide, optionallya polyacrylamide surface derived using N-(5-bromoacetamidylpentyl)acrylamide (BRAPA), preferably the polyacrylamide surface is coupled toa solid support, such as glass.

Reversible Immobilization

Synthetic polynucleotides having a predefined sequence can besynthesised in accordance with the invention using binding surfaces andstructures, such as microparticles and beads, which facilitatereversible immobilization. Solid phase reversible immobilization (SPRI)methods or modified methods are known in the art and may be employed(e.g. see DeAngelis M. M. et al. (1995) Solid-Phase ReversibleImmobilization for the Isolation of PCR Products, Nucleic AcidsResearch, 23(22): 4742-4743.).

Surfaces can be provided in the form of microparticles, such asparamagnetic beads. Paramagnetic beads can agglomerate under theinfluence of a magnetic field. For example, paramagnetic surfaces can beprovided with chemical groups, e.g. carboxyl groups, which inappropriate attachment conditions will act as binding moieties fornucleic acids, as described in more detail below. Nucleic acids can beeluted from such surfaces in appropriate elution conditions. Surfaces ofmicroparticles and beads can be provided with UV-sensitivepolycarbonate. Nucleic acids can be bound to the activated surface inthe presence of a suitable immobilization buffer.

Microparticles and beads may be allowed to move freely within a reactionsolution and then reversibly immobilized, e.g. by holding the beadwithin a microwell or pit etched into a surface. A bead can be localisedas part of an array e.g. by the use of a unique nucleic acid “barcode”attached to the bead or by the use of colour-coding.

Thus before initiating synthesis of a new double-stranded polynucleotideof predefined sequence, anchor/scaffold polynucleotides in accordancewith the invention can be synthesised and then reversibly immobilized tosuch binding surfaces. Polynucleotides synthesised by methods of theinvention can be synthesised whilst reversibly immobilized to suchbinding surfaces.

Microfluidic Techniques and Systems

The surface may be part of an electrowetting-on-dielectric system(EWOD). EWOD systems provide a dielectric-coated surface whichfacilitates microfluidic manipulation of very small liquid volumes inthe form of microdroplets (e.g. see Chou, W-L., et al. (2015) RecentAdvances in Applications of Droplet Microfluidics, Micromachines, 6:1249-1271.). Droplet volumes can programmably be created, moved,partitioned and combined on-chip by electrowetting techniques. Thuselectrowetting systems provide alternative means to reversiblyimmobilize polynucleotides during and after synthesis.

Polynucleotides having a predefined sequence may be synthesised in solidphase by methods described herein, wherein polynucleotides areimmobilized on an EWOD surface and required steps in each cyclefacilitated by electrowetting techniques. For example, in methodsinvolving scaffold polynucleotides and requiring incorporation,cleavage, ligation and deprotection steps, reagents required for eachstep, as well as for any required washing steps to remove used andunwanted reagent, can be provided in the form of microdropletstransported under the influence of an electric field via electrowettingtechniques.

Other microfluidic platforms are available which may be used in thesynthesis methods of the invention. For example, the emulsion-basedmicrodroplet techniques which are commonly employed for nucleic acidmanipulation can be used. In such systems microdroplets are formed in anemulsion created by the mixing of two immiscible fluids, typically waterand an oil. Emulsion microdroplets can be programmably be created,moved, partitioned and combined in microfluidic networks. Hydrogelsystems are also available. In any of the synthesis methods describedherein microdroplets may be manipulated in any suitable compatiblesystem, such as EWOD systems described above and other microfluidicsystems, e.g. microfluidic systems comprising architectures based oncomponents comprising elastomeric materials.

Microdroplets may be of any suitable size, provided that they arecompatible with the synthesis methods herein. Microdroplet sizes willvary depending upon the particular system employed and the relevantarchitecture of the system. Sizes may thus be adapted as appropriate. Inany of the synthesis methods described herein droplet diameters may bein the range from about 150 nm to about 5 mm. Droplet diameters below 1μm may be verified by means known in the art, such as by techniquesinvolving capillary jet methods, e.g. as described in Gañán-Calvo et al.(Nature Physics, 2007, 3, pp 737-′742)

Sequencing of Intermediate or Final Synthesis Products.

The intermediate products of synthesis or assembly, or the finalpolynucleotide synthesis products may be sequenced as a quality controlcheck to determine whether the desired polynucleotide or polynucleotideshave been correctly synthesised or assembled. The polynucleotide orpolynucleotides of interest can be removed from the solid phasesynthesis platform and sequenced by any one of a number of knowncommercially available sequencing techniques such as nanopore sequencingusing a MinION™ device sold by Oxford Nanopore Technologies Ltd. In aparticular example, the sequencing may be carried out on the solid phaseplatform itself, removing the need to transfer the polynucleotide to aseparate synthesis device. Sequencing may be conveniently carried out onthe same electrowetting device, such as an EWOD device as used forsynthesis whereby the synthesis device comprises one or more measurementelectrode pairs. A droplet comprising the polynucleotide of interest canbe contacted with one of the electrodes of the electrode pair, thedroplet forming a droplet interface bilayer with a second droplet incontact with the second electrode of the electrode pair wherein thedroplet bilayer interface comprises a nanopore in an amphipathicmembrane. The polynucleotide can be caused to translocate the nanoporefor example under enzyme control and ion current flow through thenanopore can be measured under a potential difference between theelectrode pair during passage of the polynucleotide through thenanopore. The ion current measurements over time can be recorded andused to determine the polynucleotide sequence. Prior to sequencing, thepolynucleotide may be subjected to one or more sample preparation stepsin order to optimise it for sequencing such as disclosed in patentapplication no. PCT/GB2015/050140. Examples of enzymes, amphipathicmembranes and nanopores which may be suitably employed are disclosed inpatent application nos. PCT/GB2013/052767 and PCT/GB2014/052736. Thenecessary reagents for sample preparation of the polynucleotide,nanopores, amphipathic membranes and so on may be supplied to the EWODdevice via sample inlet ports. The sample inlet ports may be connectedto reagent chambers.

Surface Attachment Chemistries

Although oligonucleotides will typically be attached chemically, theymay also be attached to surfaces by indirect means such as via affinityinteractions. For example, oligonucleotides may be functionalised withbiotin and bound to surfaces coated with avidin or streptavidin.

For the immobilization of polynucleotides to surfaces (e.g. planarsurfaces), microparticles and beads etc., a variety of surfaceattachment methods and chemistries are available. Surfaces may befunctionalised or derivatized to facilitate attachment. Suchfunctionalisations are known in the art. For example, a surface may befunctionalised with a polyhistidine-tag (hexa histidine-tag, 6×His-tag,His6 tag or His-tag®), Ni-NTA, streptavidin, biotin, an oligonucleotide,a polynucleotide (such as DNA, RNA, PNA, GNA, TNA or LNA), carboxylgroups, quaternary amine groups, thiol groups, azide groups, alkynegroups, DIBO, lipid, FLAG-tag (FLAG octapeptide), polynucleotide bindingproteins, peptides, proteins, antibodies or antibody fragments. Thesurface may be functionalised with a molecule or group whichspecifically binds to the anchor/scaffold polynucleotide.

Some examples of chemistries suitable for attaching polynucleotides tosurfaces are shown in FIG. 11i and FIG. 11 j.

In any of the methods described herein the scaffold polynucleotidecomprising the synthesis strand comprising the primer strand portion andthe portion of the support strand hybridized thereto may be tethered toa common surface via one or more covalent bonds. The one or morecovalent bonds may be formed between a functional group on the commonsurface and a functional group on the scaffold molecule. The functionalgroup on the scaffold molecule may be e.g. an amine group, a thiolgroup, a thiophosphate group or a thioamide group. The functional groupon the common surface may be a bromoacetyl group, optionally wherein thebromoacetyl group is provided on a polyacrylamide surface derived usingN-(5-bromoacetamidylpentyl) acrylamide (BRAPA).

In any of the methods of the invention a scaffold polynucleotide may beattached to a surface, either directly or indirectly, via a linker. Anysuitable linker which is biocompatible and hydrophilic in nature may beused.

A linker may be a linear linker or a branched linker.

A linker may comprise a hydrocarbon chain. A hydrocarbon chain maycomprise from 2 to about 2000 or more carbon atoms. The hydrocarbonchain may comprise an alkylene group, e.g. C2 to about 2000 or morealkylene groups. The hydrocarbon chain may have a general formula of—(CH₂)_(n)— wherein n is from 2 to about 2000 or more. The hydrocarbonchain may be optionally interrupted by one or more ester groups (i.e.—C(O)—O—) or one or more amide groups (i.e. —C(O)—N(H)—).

Any linker may be used selected from the group comprising PEG,polyacrylamide, poly(2-hydroxyethyl methacrylate),Poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers, e.g.poly(carboxybetaine methacrylate) (PCBMA),poly[N-(3-sulfopropyl)-N-methacryloxyethyl-N, N dimethyl ammoniumbetaine] (PSBMA), glycopolymers, and polypeptides.

A linker may comprise a polyethylene glycol (PEG) having a generalformula of —(CH₂—CH₂—O)n−, wherein n is from 1 to about 600 or more.

A linker may comprise oligoethylene glycol-phosphate units having ageneral formula of —[(CH₂—CH₂—O)_(n)—PO₂ ⁻—O]_(m)— where n is from 1 toabout 600 or more and m could be 1-200 or more.

Any of the above-described linkers may be attached at one end of thelinker to a scaffold molecule as described herein, and at the other endof the linker to a first functional group wherein the first functionalgroup may provide a covalent attachment to a surface. The firstfunctional group may be e.g. an amine group, a thiol group, athiophosphate group or a thioamide group as further described herein.The surface may be functionalised with a further functional group toprovide a covalent bond with the first functional group. The furtherfunctional group may be e.g. a 2-bromoacetamido group as furtherdescribed herein. Optionally a bromoacetyl group is provided on apolyacrylamide surface derived using N-(5-bromoacetamidylpentyl)acrylamide (BRAPA). The further functional group on the surface may be abromoacetyl group, optionally wherein the bromoacetyl group is providedon a polyacrylamide surface derived using N-(5-bromoacetamidylpentyl)acrylamide (BRAPA) and the first functional group may be e.g. an aminegroup, a thiol group, a thiophosphate group or a thioamide group asappropriate. The surface to which polynucleotides are attached maycomprise a gel. The surface comprises a polyacrylamide surface, such asabout 2% polyacrylamide, preferably the polyacrylamide surface iscoupled to a solid support such as glass.

In any of the methods of the invention a scaffold polynucleotide mayoptionally be attached to a linker via a branching nucleotideincorporated into the scaffold polynucleotide. Any suitable branchingnucleotide may be used with any suitable compatible linker.

Prior to initiating synthesis cycles of the invention, scaffoldpolynucleotides may be synthesised with one or more branchingnucleotides incorporated into the scaffold polynucleotide. The exactposition at which the one or more branching nucleotides are incorporatedinto the scaffold polynucleotide, and thus where a linker may beattached, may vary and may be chosen as desired. The position may e.g.be at the terminal end of a support strand and/or a synthesis strand ore.g. in the loop region which connects the support strand to thesynthesis strand in embodiments which comprise a hairpin loop.

During synthesis of the scaffold polynucleotide the one or morebranching nucleotides may be incorporated into the scaffoldpolynucleotide with a blocking group which blocks a reactive group ofthe branching moiety. The blocking group may then be removed (deblocked)prior to the coupling to the branching moiety of the linker, or a firstunit (molecule) of the linker if a linker comprises multiple units.

During synthesis of the scaffold polynucleotide the one or morebranching nucleotides may be incorporated into the scaffoldpolynucleotide with a group suitable for use in a subsequent “clickchemistry” reaction to couple to the branching moiety the linker, or afirst unit of the linker if a linker comprises multiple units. Anexample of such a group is an acetylene group.

Some non-limiting exemplary branching nucleotides are shown below.

A linker may optionally comprise one or more spacer molecules (units),such as e.g. an Sp9 spacer, wherein the first spacer unit is attached tothe branching nucleotide.

The linker may comprise one or more further spacer groups attached tothe first spacer group. For example, the linker may comprise multiplee.g. Sp9 spacer groups. A first spacer group is attached to thebranching moiety and then one or more further spacer groups aresequentially added to extend a spacer chain comprising multiple spacerunits connected with phosphate groups therebetween.

Shown below are some non-limiting examples of spacer units (Sp3, Sp9 andSp13) which could comprise the first spacer unit attached to a branchingnucleotide, or a further spacer unit to be attached to an existingspacer unit already attached to the branching nucleotide.

A linker may comprise one or more ethylene glycol units.

A linker may comprise an oligonucleotide, wherein multiple units arenucleotides.

In the structures depicted above the term 5″ is used to differentiatefrom the 5′ end of the nucleotide to which the branching moiety isattached, wherein 5′ has its ordinary meaning in the art. By 5″ it isintended to mean a position on the nucleotide from which a linker can beextended. The 5″ position may vary. The 5″ position is typically aposition in the nucleobase of the nucleotide. The 5″ position in thenucleobase may vary depending on the nature of the desired branchingmoiety, as depicted in the structures above.

Microarrays

Any of the polynucleotide synthesis methods described herein may be usedto manufacture a polynucleotide microarray (Trevino, V. et al., Mol.Med. 2007 13, pp 527-541). Thus anchor or scaffold polynucleotides maybe tethered to a plurality of individually addressable reaction sites ona surface and polynucleotides having a predefined sequence may besynthesised in situ on the microarray.

Following synthesis, at each reaction area the polynucleotide ofpredefined sequence may be provided with a unique sequence. The anchoror scaffold polynucleotides may be provided with barcode sequences tofacilitate identification.

Other than the method of synthesising the polynucleotides of predefinedsequence, microarray manufacture may be performed using techniquescommonly used in this technical field, including techniques describedherein. For example, anchor or scaffold polynucleotides may be tetheredto surfaces using known surface attachment methods and chemistries,including those described herein.

Following synthesis of the polynucleotides of predefined sequence, theremay be provided a final cleavage step to remove any unwantedpolynucleotide sequence from untethered ends.

Polynucleotides of predefined sequence may be provided at reaction sitesin double-stranded form. Alternatively, following synthesisdouble-stranded polynucleotides may be separated and one strand removed,leaving single-stranded polynucleotides at reaction sites. Selectivetethering of strands may be provided to facilitate this process. Forexample, in methods involving a scaffold polynucleotide the synthesisstrand may be tethered to a surface and the support strand may beuntethered, or vice versa. The synthesis strand may be provided with anon-cleavable linker and the support strand may be provided with acleavable linker, or vice versa. Separation of strands may be performedby conventional methods, such as heat treatment.

Assembly of Synthetic Polynucleotides

A polynucleotide having a predefined sequence synthesised by methodsdescribed herein, and optionally amplified by methods described herein,may be joined to one or more other such polynucleotides to create largersynthetic polynucleotides.

Joining of multiple polynucleotides can be achieved by techniquescommonly known in the art. A first polynucleotide and one or moreadditional polynucleotides synthesised by methods described herein maybe cleaved to create compatible termini and then polynucleotides joinedtogether by ligation. Cleavage can be achieved by any suitable means.Typically, restriction enzyme cleavage sites may be created inpolynucleotides and then restriction enzymes used to perform thecleavage step, thus releasing the synthesised polynucleotides from anyanchor/scaffold polynucleotide. Cleavage sites could be designed as partof the anchor/scaffold polynucleotides. Alternatively, cleavage sitescould be created within the newly-synthesised polynucleotide as part ofthe predefined nucleotide sequence.

Assembly of polynucleotides is preferably performed using solid phasemethods. For example, following synthesis a first polynucleotide may besubject to a single cleavage at a suitable position distal to the siteof surface immobilization. The first polynucleotide will thus remainimmobilized to the surface, and the single cleavage will generate aterminus compatible for joining to another polynucleotide. An additionalpolynucleotide may be subject to cleavage at two suitable positions togenerate at each terminus a compatible end for joining to otherpolynucleotides, and at the same time releasing the additionalpolynucleotide from surface immobilization. The additionalpolynucleotide may be compatibly joined with the first polynucleotidethus creating a larger immobilized polynucleotide having a predefinedsequence and having a terminus compatible for joining to yet anotheradditional polynucleotide. Thus iterative cycles of joining ofpreselected cleaved synthetic polynucleotides may create much longersynthetic polynucleotide molecules. The order of joining of theadditional polynucleotides will be determined by the required predefinedsequence.

Thus the assembly methods of the invention may allow the creation ofsynthetic polynucleotide molecules having lengths in the order of one ormore Mb.

The assembly and/or synthesis methods of the invention may be performedusing apparatuses known in the art. Techniques and apparatuses areavailable which allow very small volumes of reagents to be selectivelymoved, partitioned and combined with other volumes in differentlocations of an array, typically in the form of droplets Electrowettingtechniques, such as electrowetting-on-dielectric (EWOD), may beemployed, as described above. Suitable electrowetting techniques andsystems that may be employed in the invention that are able tomanipulate droplets are disclosed for example in U.S. Pat. Nos.8,653,832, 8,828,336, US20140197028 and US20140202863.

Cleavage from the solid phase may be achieved by providing cleavablelinkers in one or both the primer strand portion and the portion of thesupport strand hybridized thereto. The cleavable linker may be e.g. a UVcleavable linker.

Examples of cleavage methods involving enzymatic cleavage are shown inFIG. 29. The schematic shows a scaffold polynucleotide attached to asurface (via black diamond structures) and comprising a polynucleotideof predefined sequence. The scaffold polynucleotide comprises top andbottom hairpins. In each case the top hairpin can be cleaved using acleavage step utilizing the universal nucleotide to define a cleavagesite. The bottom hairpin can be removed by a restriction endonucleasevia a site that is engineered into the scaffold polynucleotide orengineered into the newly-synthesised polynucleotide of predefinedsequence.

Thus polynucleotides having a predefined sequence may be synthesisedwhilst immobilized to an electrowetting surface, as described above.Synthesised polynucleotides may be cleaved from the electrowettingsurface and moved under the influence of an electric field in the formof a droplet. Droplets may be combined at specific reaction sites on thesurface where they may deliver cleaved synthesised polynucleotides forligation with other cleaved synthesised polynucleotides. Polynucleotidescan then be joined, for example by ligation. Using such techniquespopulations of different polynucleotides may be synthesised and attachedin order according to the predefined sequence desired. Using suchsystems a fully automated polynucleotide synthesis and assembly systemmay be designed. The system may be programmed to receive a desiredsequence, supply reagents, perform synthesis cycles and subsequentlyassemble desired polynucleotides according to the predefined sequencedesired.

Systems and Kits

The invention also provides polynucleotide synthesis systems forcarrying out any of the synthesis methods described and defined herein,as well as any of the subsequent amplification and assembly stepsdescribed and defined herein.

Typically, synthesis cycle reactions will be carried out byincorporating nucleotides of predefined sequence into scaffoldpolynucleotide molecules which are tethered to a surface by meansdescribed and defined herein. The surface may be any suitable surface asdescribed and defined herein.

In one embodiment, reactions to incorporate nucleotides of predefinedsequence into a scaffold polynucleotide molecule involve performing anyof the synthesis methods on a scaffold polynucleotide within a reactionarea.

A reaction area is any area of a suitable substrate to which a scaffoldpolynucleotide molecule is attached and wherein reagents for performingthe synthesis methods may be delivered.

In one embodiment a reaction area may be a single area of a surfacecomprising a single scaffold polynucleotide molecule wherein the singlescaffold polynucleotide molecule can be addressed with reagents.

In another embodiment a reaction area may be a single area of a surfacecomprising multiple scaffold polynucleotide molecules, wherein thescaffold polynucleotide molecules cannot be individually addressed withreagent in isolation from each other. Thus in such an embodiment themultiple scaffold polynucleotide molecules in the reaction area areexposed to the same reagents and conditions and may thus give rise tosynthetic polynucleotide molecules having the same or substantially thesame nucleotide sequence.

In one embodiment a synthesis system for carrying out any of thesynthesis methods described and defined herein may comprise multiplereaction areas, wherein each reaction area comprises one or moreattached scaffold polynucleotide molecules and wherein each reactionarea may be individually addressed with reagent in isolation from eachof the other reaction areas. Such a system may be configured e.g. in theform of an array, e.g. wherein reaction areas are formed upon asubstrate, typically a planar substrate.

A system having a substrate comprising a single reaction area orcomprising multiple reaction areas may be comprised within e.g. an EWODsystem or a microfluidic system and the systems configured to deliverreagents to the reaction site. EWOD and microfluidic systems aredescribed in more detail herein. For example an EWOD system may beconfigured to deliver reagents to the reaction site(s) under electricalcontrol. A microfluidic system, such as comprising microfabricatedarchitecture e.g. as formed from elastomeric or similar material, may beconfigured to deliver reagents to the reaction site(s) under fluidicpressure and/or suction control or by mechanical means. Reagents may bedelivered by any suitable means, for example via carbon nanotubes actingas conduits for reagent delivery. Any suitable system may be envisaged.

EWOD, microfluidic and other systems may be configured to deliver anyother desired reagents to reaction sites, such as enzymes for cleaving asynthesised double-stranded polynucleotide from the scaffoldpolynucleotide following synthesis, and/or reagents for cleaving alinker to release an entire scaffold polynucleotide from the substrateand/or reagents for amplifying a polynucleotide molecule followingsynthesis or any region or portion thereof, and/or reagents forassembling larger polynucleotide molecules from smaller polynucleotidemolecules which have been synthesised according to the synthesis methodsof the invention.

The invention also provides kits for carrying out any of the synthesismethods described and defined herein. A kit may contain any desiredcombination of reagents for performing any of the synthesis and/orassembly methods of the invention described and defined herein. Forexample, a kit may comprise any one or more volume(s) of reactionreagents comprising scaffold polynucleotides, volume(s) of reactionreagents corresponding to any one or more steps of the synthesis cyclesdescribed and defined herein, volume(s) of reaction reagents comprisingnucleotides comprising reversible blocking groups or reversibleterminator groups, volume(s) of reaction reagents for amplifying one ormore polynucleotide molecules following synthesis or any region orportion thereof, volume(s) of reaction reagents for assembling largerpolynucleotide molecules from smaller polynucleotide molecules whichhave been synthesised according to the synthesis methods of theinvention, volume(s) of reaction reagents for cleaving a synthesiseddouble-stranded polynucleotide from the scaffold polynucleotidefollowing synthesis, and volume(s) of reaction reagents for cleaving oneor more linkers to release entire scaffold polynucleotides from asubstrate.

Synthesis Strand

In methods of synthesising a polynucleotide or oligonucleotide describedherein including, but not limited to, synthesis method versions 1 to 5of the invention as described in FIGS. 1 to 5 and further herein, thescaffold polynucleotide is provided with a synthesis strand. Duringcycles of synthesis each new nucleotide of the predefined sequence isincorporated into the synthesis strand. An enzyme, such as a polymeraseenzyme or enzyme having terminal transferase activity, can be used tocatalyse incorporation/addition of each new nucleotide, nucleotideanalogue/derivative or non-nucleotide. The synthesis strand comprises aprimer strand portion and preferably comprises a helper strand portion.

Helper Strand

A helper strand may be provided in the scaffold polynucleotide tofacilitate binding of cleavage enzyme(s) at the cleavage step. A helperstrand may be provided in the ligation polynucleotide to facilitateligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide at the ligation step. The helper strand may be omitted,provided that alternative means are provided to ensure binding ofcleavage enzyme(s) at the cleavage step and to ensure ligation at theligation step, if necessary. In preferred methods of the invention thesynthesis strand is provided with a helper strand. Preferably, theligation polynucleotide is provided with a helper strand and the helperstrand is retained in the scaffold polynucleotide at the cleavage step,as show in in FIGS. 1 to 5.

There are no special requirements for the parameters of length, sequenceand structure of the helper strand, provided that the helper strand issuitable to facilitate binding of cleavage enzyme(s) at the cleavagestep.

The helper strand may comprise nucleotides, nucleotideanalogues/derivatives and/or non-nucleotides.

Preferably, within the region of sequence of the helper strandmismatches with the support strand should be avoided, GC- and AT-richregions should be avoided, and in addition regions of secondarystructure such as hairpins or bulges should be avoided.

The length of the helper strand may be 10 bases or more. Optionally, thelength of the helper strand may be 15 bases or more, preferably 30 basesor more. However, the length of the helper strand may be varied,provided that the helper strand is capable of facilitating cleavageand/or ligation.

The helper strand must be hybridized to the corresponding region of thesupport strand. It is not essential that the entirety of the helperstrand is hybridized to the corresponding region of the support strand,provided that the helper strand can facilitate binding of cleavageenzyme(s) at the cleavage step and/or binding of ligase enzyme at theligation step. Thus, mismatches between the helper strand and thecorresponding region of the support strand can be tolerated. The helperstrand may be longer than the corresponding region of the supportstrand. The support strand may extend beyond the region whichcorresponds with the helper strand in the direction distal to the primerstrand. The helper strand may be connected to the corresponding regionof the support strand, e.g. via a hairpin.

The helper strand is preferably hybridized to the support strand suchthat the terminal nucleotide of the helper strand at the site of thenick occupies the next sequential nucleotide position in the synthesisstrand relative to the terminal nucleotide of the primer strand at thesite of the nick. Thus in this configuration there are no nucleotideposition gaps between the helper strand and the primer strand. Thehelper strand and primer strand will nevertheless be physicallyseparated due to the presence of the single-stranded break or nick.Preferably, the terminal nucleobase of the helper strand at the site ofthe nick is hybridized to its partner nucleotide in the support strand.

The nucleotide in the helper strand which pairs with the universalnucleotide may be any suitable nucleotide. Preferably, pairings whichare likely to distort the helical structure of the molecule should beavoided. Preferably cytosine acts as a partner for the universalnucleotide. In a particularly preferred embodiment the universalnucleotide is inosine, or an analogue, variant or derivative thereof,and the partner nucleotide for the universal nucleotide in the helperstrand is cytosine.

Removal of Helper Strand

Although it is preferred that the helper strand is retained during stepsof synthesis, in any of the synthesis methods of the invention describedherein, including exemplary method versions 1 to 5, in step (1) (i.e. inthe first cycle) of providing a scaffold polynucleotide comprising asynthesis strand and a support strand hybridized thereto (101, 201, 301,401, 501), the synthesis strand may be provided without a helper strand.

Furthermore, in any one or more cycles of synthesis, or in all cycles ofsynthesis, after the step of ligating the double-stranded ligationpolynucleotide to the cleaved scaffold polynucleotide and before thestep of cleavage of the scaffold polynucleotide (106, 206, 306, 406,506), the helper strand portion of the synthesis strand may be removedfrom the scaffold polynucleotide.

The helper strand portion of the synthesis strand may be removed fromthe scaffold polynucleotide by any suitable means including, but notlimited to: (i) heating the scaffold polynucleotide to a temperature ofabout 80° C. to about 95° C. and separating the helper strand portionfrom the scaffold polynucleotide, (ii) treating the scaffoldpolynucleotide with urea solution, such as 8M urea and separating thehelper strand portion from the scaffold polynucleotide, (iii) treatingthe scaffold polynucleotide with formamide or formamide solution, suchas 100% formamide and separating the helper strand portion from thescaffold polynucleotide, or (iv) contacting the scaffold polynucleotidewith a single-stranded polynucleotide molecule which comprises a regionof nucleotide sequence which is complementary with the sequence of thehelper strand portion, thereby competitively inhibiting thehybridisation of the helper strand portion to the scaffoldpolynucleotide.

In methods wherein the helper strand portion is removed from thescaffold polynucleotide after the step of ligating the double-strandedligation polynucleotide to the cleaved scaffold polynucleotide andbefore the step of cleavage of the scaffold polynucleotide, the cleavagestep will comprise cleaving the support strand in the absence of adouble-stranded region provided by the helper strand. Any suitableenzyme may be chosen for performing such a cleavage step, such asselected from any suitable enzyme disclosed herein.

Primer Strand

The primer strand portion should be suitable to allow an enzyme, such asa polymerase enzyme or enzyme having terminal transferase activity, toinitiate synthesis, i.e. catalyse the addition of a new nucleotide atthe terminal end of the primer strand at the site of the nick.

The primer strand may comprise a region of sequence which can act toprime new polynucleotide synthesis (e.g. as shown by the dotted line inthe structures depicted in each of FIGS. 1 to 5). The primer strand mayconsist of a region of sequence which can act to prime newpolynucleotide synthesis, thus the entirety of the primer strand may besequence which can act to prime new polynucleotide synthesis asdescribed herein.

There are no special requirements for the parameters of length, sequenceand structure of the primer strand, provided that the primer strand issuitable to prime new polynucleotide synthesis.

The primer strand may comprise nucleotides, nucleotideanalogues/derivatives and/or non-nucleotides.

The skilled person is readily able to construct a primer strand whichwill be capable of priming new polynucleotide synthesis. Thus, withinthe region of sequence of the primer strand which can act to prime newpolynucleotide synthesis mismatches with the support strand should beavoided, GC- and AT-rich regions should be avoided, and in additionregions of secondary structure such as hairpins or bulges should beavoided.

The length of the region of sequence of the primer strand which can actto prime new polynucleotide synthesis can be chosen by the skilledperson depending on preference and the polymerase enzyme to be used. Thelength of this region may be 7 bases or more, 8 bases or more, 9 basesor more or 10 bases or more. Optionally the length of this region willbe 15 bases or more, preferably 30 bases or more.

The primer strand must be hybridized to the corresponding region of thesupport strand. It is not essential that the entirety of the primerstrand is hybridized to the corresponding region of the support strand,provided that the primer strand is capable of priming new polynucleotidesynthesis. Thus, mismatches between the primer strand and thecorresponding region of the support strand can be tolerated to a degree.Preferably, the region of sequence of the primer strand which can act toprime new polynucleotide synthesis should comprise nucleobases which arecomplementary to corresponding nucleobases in the support strand.

The primer strand may be longer than the corresponding region of thesupport strand. The support strand may extend beyond the region whichcorresponds with the primer strand in the direction distal to the helperstrand. The primer strand may be connected to the corresponding regionof the support strand, e.g. via a hairpin.

Support Strand

In methods of the invention including, but not limited to, synthesismethod versions of the invention 1 to 5, as described above, thescaffold polynucleotide is provided with a support strand. The supportstrand is hybridized to the synthesis strand. There are no specialrequirements for the parameters of length, sequence and structure of thesupport strand, provided that the support strand is compatible with theprimer strand portion and, if included, the helper strand portion of thesynthesis strand, as described above.

RNA Synthesis

Methods described for DNA synthesis may be adapted for the synthesis ofRNA. In one adaptation the synthesis steps described for synthesismethod versions of the invention 1 to 5 may be adapted. Thus in each ofsynthesis method versions 1 to 5 the support strand of the scaffoldpolynucleotide is a DNA strand, as described above. The primer strandportion of the synthesis strand of the scaffold polynucleotide is an RNAstrand. The helper strand, if present, is preferably an RNA strand. Thehelper strand, if present, may be a DNA strand.

Nucleotides may be incorporated from ribonucleoside-5′-O-triphosphates(NTPs) which may be modified to comprise a reversible terminator group,as described above. Preferably3′-O-modified-ribonucleoside-5′-O-triphosphates are used. Modifiednucleotides are incorporated by the action of RNA polymerase.

Thus the descriptions relating to synthesis method versions of theinvention 1 to 5 may be applied mutatis mutandis for RNA synthesis butadapted as described.

FIGS. 31 and 32 describe reaction schemes for RNA synthesis which areadaptations of DNA synthesis method versions 1, 2 of the Examples asdepicted in FIGS. 6 and 7 respectively. Method versions of the invention1 to 5, as depicted in FIGS. 1 to 5 respectively may be adapted in thesame way.

In any of the adapted methods for RNA synthesis, the above descriptionsof support strand, primer strand, helper strand, ligation polynucleotideand universal nucleotide may be applied mutatis mutandis but adapted asdescribed. Cleavage steps and cleavage positions as previously describedmay be applied mutatis mutandis since the support strand which comprisesthe universal nucleotide is a DNA strand. In a preferred embodimentSplintR DNA ligase is used in the ligation step.

Exemplary Methods

Exemplary methods of synthesising a polynucleotide or an oligonucleotidemolecule according to the invention are described herein, including inthe appended claims.

In the following five exemplary methods of synthesising a polynucleotideor an oligonucleotide molecule according to the invention references tosynthesis method versions 1, 2, 3, 4 and 5 are to be interpretedaccording to the reaction schematics set out respectively in FIGS. 1, 2,3, 4 and 5 and not according to the reaction schematics set out in anyof FIGS. 6 to 10 or descriptions of the same in the Examples section.The reaction schematics set out in any of FIGS. 6 to 10 and descriptionsof the same in the Examples section below provide illustrative supportfor the methods of the invention based on reaction schemes which aremodified in comparison with the methods of the present invention. Thusreference signs in the text below correspond with those in FIGS. 1 to 5.

In each exemplary method described below, the structures described ineach step may be referred to by reference to specific figures with theaid of reference signs as appropriate. However, such references are notintended to be limited to the structures shown in the figures, and thedescription of the relevant structures correspond to the descriptionthereof as provided herein in its entirety, including but not limited tothose specifically illustrated.

Five non-limiting exemplary methods, method versions 1 to 5, aredescribed below (see e.g. FIGS. 1 to 5 respectively). In step (1) ofeach of these exemplary methods a scaffold polynucleotide (see structuredepicted in step 1 of each of FIGS. 1 to 5) is provided (101, 201, 301,401, 501) comprising a synthesis strand (see strand labelled “b” in thestructure depicted in step 1 of each of FIGS. 1 to 5) hybridized to acomplementary support strand (see strand labelled “a” in structuredepicted in step 1 of each of FIGS. 1 to 5).

The scaffold polynucleotide is double-stranded and provides a supportstructure to accommodate the region of synthetic polynucleotide as it issynthesised de novo. The scaffold polynucleotide comprises a synthesisstrand comprising a primer strand portion (see dotted portion of strandlabelled “b” in structure depicted in step 1 of each of FIGS. 1 to 5)and a helper strand portion (see dashed portion of strand labelled “b”in structure depicted in step 1 of each of FIGS. 1 to 5) separated by asingle-strand break or “nick”. As described in more detail herein, incertain of the exemplary method versions 1 to 5 and variants thereofdescribed herein the helper strand may be removed prior to the cleavagestep (2), e.g. by denaturation.

Both the primer strand portion and the helper strand portion of thesynthesis strand are provided hybridized to a complementary supportstrand.

In each of the five methods a universal nucleotide (labelled “Un” in thestructures depicted in each of FIGS. 1 to 5) is provided in the supportstrand which facilitates cleavage of the scaffold polynucleotide (102,202, 302, 402, 502). The role of the universal nucleotide will beapparent from the detailed description of each method below.

Cleavage of the scaffold polynucleotide (step 2) results in loss of thehelper strand, if present immediately prior to cleavage, and loss of thesupport strand comprising the universal nucleotide. Cleavage leaves inplace a cleaved double-stranded scaffold polynucleotide comprising, atthe site of cleavage, a cleaved terminal end of the support strand andthe terminal end of the primer strand portion of the synthesis strandwhich comprised the nick site prior to cleavage.

The terminal end of the primer strand portion of the synthesis strandprovides a primer site for use in the initiation of synthesis by anenzyme having transferase activity. Thus the enzyme will act to extendthe terminal nucleotide of the primer strand portion. This terminalnucleotide will therefore typically define a 3′ terminus of the primerstrand portion, e.g. to allow extension by polymerase or transferaseenzymes which catalyse extension in a 5′ to 3′ direction. The oppositeterminus of the synthesis strand comprising the primer strand portionwill consequently typically define a 5′ terminus of the synthesisstrand, and the terminal nucleotide of the support strand adjacent the5′ terminus of the synthesis strand will consequently typically definethe 3′ terminus of the support strand.

The terminal nucleotide of the helper strand portion of the synthesisstrand, which is positioned at the site of the single-strand break, willtypically define a 5′ terminus of the helper strand portion andconsequently the opposite terminus of the helper strand portion of thesynthesis strand will typically define the 3′ terminus of the synthesisstrand.

The single-stranded break or “nick” between the helper strand portionand the primer strand portion of the synthesis strand is typicallyachieved by providing the (5′) terminal nucleotide of the helper strandin such a form that will prevent it from ligating to the primer strandat the ligation step. This can typically be achieved by providing the(5′) terminal nucleotide of the helper strand without a phosphate group.The break is typically achieved by assembling the scaffoldpolynucleotide from separate components comprising: (i) the supportstrand; (ii) the helper strand portion of the synthesis strand,typically having a non-phosphorylated (5′) terminal nucleotide; and(iii) the synthesis strand portion comprising the primer strand portion.Upon mixing of the components in suitable conditions the scaffoldpolynucleotide forms upon hybridization of the separate components.

In certain methods described herein the helper strand may optionally beremoved from the scaffold polynucleotide prior to the step of cleavage,e.g. by denaturation and release from the support strand to which it waspreviously hybridized.

Cleavage results in removal of the support strand comprising theuniversal nucleotide and the helper strand hybridised to the supportstrand, if present immediately prior to cleavage.

In step (3) of the methods a first nucleotide in the predefinednucleotide sequence is incorporated into the synthesis strand by theaction of an enzyme having transferase activity such as polymerase or aterminal nucleotidyl transferase enzyme (102, 202, 302, 402, 502). Thefirst nucleotide is provided with a reversible terminator group(depicted as the small triangle of the incorporated nucleotide in step 3of each of FIGS. 1 to 5) which prevents further extension by the enzyme.Thus in step (3) only a single nucleotide is incorporated.

Nucleotides comprising any suitable reversible terminator group could beused. Preferred nucleotides with reversible terminator groups are3′-O-allyl-dNTPs and/or 3′-O-azidomethyl-dNTPs as described herein.

In step (4) of the methods a deprotection step is performed to removethe reversible terminator group from the incorporated first nucleotideof the predefined nucleotide sequence (104, 204, 304, 404, 504). Thedeprotection step may alternatively be performed after the ligation step(5) described below.

In step (5) of the methods a ligation step is performed (105, 205, 305,405, 505) wherein a ligation polynucleotide is ligated to the cleaveddouble-stranded scaffold polynucleotide comprising the first nucleotideof the predefined nucleotide sequence. The ligation polynucleotidecomprises a support strand and a helper strand hybridized to the supportstrand. The ligation polynucleotide comprises a complementary ligationend which is complementary with the cleaved end of the cleaveddouble-stranded scaffold polynucleotide comprising the first nucleotide.The support strand of the ligation polynucleotide comprises a partnernucleotide for the first nucleotide of the predefined nucleotidesequence at the complementary ligation end wherein upon ligation thepartner nucleotide is positioned opposite the first nucleotide of thepredefined nucleotide sequence to form a nucleotide pair. The supportstrand of the ligation polynucleotide also comprises a universalnucleotide at the complementary ligation end which will facilitatecleavage in the next cycle of synthesis. The terminal nucleotide of thehelper strand of the ligation polynucleotide at the complementaryligation end is provided such that the helper strand cannot be ligatedto the primer strand. This is typically achieved by providing theterminal nucleotide of the helper strand without a phosphate group. Thusupon ligation of the support strand of the ligation polynucleotide andthe support strand of the cleaved double-stranded scaffoldpolynucleotide, a single-strand break or “nick” is provided in thesynthesis strand between the primer strand portion and the helperstrand.

Upon ligation of the ligation polynucleotide to the cleaveddouble-stranded scaffold polynucleotide an intact double-strandedscaffold polynucleotide is reformed comprising a newly incorporatednucleotide pair and a universal nucleotide for use in facilitatingcleavage in the next cycle of synthesis.

Iterative cycles of synthesis comprising the same steps as describedabove are performed in order to generate the synthetic polynucleotide.

Specific methods are described in more detail below.

Synthesis Method Version 1

In a first exemplary version of the synthesis method of the invention adouble-stranded scaffold polynucleotide is provided comprising auniversal nucleotide positioned in the support strand (step 1 of FIG. 1;101). In each cycle of synthesis the scaffold polynucleotide is cleavedat a cleavage site defined by a sequence comprising the universalnucleotide (step 2 of FIG. 1; 102, 107).

Cleavage of the scaffold polynucleotide (step 2) results in loss of thehelper strand, if present immediately prior to cleavage, and loss of thesupport strand comprising the universal nucleotide. Cleavage leaves inplace a cleaved double-stranded scaffold polynucleotide comprising, atthe site of cleavage, a cleaved terminal end of the support strand andthe terminal end of the primer strand portion of the synthesis strandwhich comprised the nick site prior to cleavage. Cleavage results in ablunt-ended cleaved double-stranded scaffold polynucleotide at the siteof cleavage, with no overhang.

In step (3) of the methods a first nucleotide in the predefinednucleotide sequence is added to the terminal end of the primer strandportion of the synthesis strand by the action of an enzyme havingtransferase activity such as polymerase or a terminal nucleotidyltransferase enzyme (103). The first nucleotide is provided with areversible terminator group which prevents further extension by theenzyme. Thus in step (3) only a single nucleotide is incorporated.

In step (4) of the methods a deprotection step is performed to removethe terminator group from the newly-incorporated nucleotide. Inprinciple the deprotection step may be performed after the ligation step(5). Performance of the deprotection step as step (4) is preferred.

In step (5) of the methods a ligation polynucleotide (see structuredepicted at the top left of the upper part of FIG. 1) is ligated to thecleaved scaffold polynucleotide. Ligation incorporates a partnernucleotide into the scaffold polynucleotide and allows thenewly-incorporated nucleotide to pair with the partner nucleotide (step5 of FIG. 1; 105), thus completing a synthesis cycle.

In the first exemplary version of the synthesis method of the inventiona scaffold polynucleotide is provided in step (1) (101) as describedabove. In this method the universal nucleotide in the support strand ofthe scaffold polynucleotide is positioned opposite the terminalnucleotide of the helper strand at the single-strand break site(labelled “X” in the structures of FIG. 1), and is paired therewith (seestructure depicted in step 1 of FIG. 1).

In step (2) of the method the scaffold polynucleotide is cleaved (102)at a cleavage site. The cleavage site is defined by a sequencecomprising the universal nucleotide in the support strand. Cleavageresults in a double-stranded break in the scaffold polynucleotide.Cleavage of the scaffold polynucleotide (step 2) results in loss of thehelper strand, if present immediately prior to cleavage, and loss of thesupport strand comprising the universal nucleotide. Cleavage of thescaffold polynucleotide leaves in place a cleaved double-strandedscaffold polynucleotide comprising, at the site of cleavage, a cleavedterminal end of the support strand and the terminal end of the primerstrand portion of the synthesis strand which comprised the nick siteprior to cleavage.

The synthesis strand is already provided with a single-stranded break or“nick” in this exemplary method, thus only cleavage of the supportstrand is necessary to provide a double-stranded break in the scaffoldpolynucleotide.

In this exemplary method version cleavage generates a blunt-endedcleaved double-stranded scaffold polynucleotide with no overhang ineither the synthesis strand or the support strand.

In this method the universal nucleotide occupies position “n” in thesupport strand prior to the cleavage step. To obtain such a blunt-endedcleaved double-stranded scaffold polynucleotide when the universalnucleotide occupies position n in the support strand, the support strandis cleaved at a specific position relative to the universal nucleotide.The support strand of the scaffold polynucleotide is cleaved betweennucleotide positions n and n−1.

By “n” it is meant the nucleotide position in the support strand whichis opposite the position in the synthesis strand which will be occupiedby the nucleotide of the predefined sequence upon its addition to theterminal end of the cleaved synthesis strand in that cycle of synthesis.By “n” it is also meant the nucleotide position in the synthesis strandwhich will be occupied by the nucleotide of the predefined sequence uponits addition to the terminal end of the primer strand portion of thesynthesis strand in that cycle of synthesis. Thus at the cleavage step,the universal nucleotide at position n in the support strand is oppositethe position which will be occupied by the nucleotide of the predefinedsequence incorporated in that cycle of synthesis. At steps (1) and (2)the position which will be occupied by the nucleotide of the predefinedsequence incorporated in that cycle of synthesis corresponds with theterminal nucleotide of the helper strand portion of the synthesis strand(shown as “X” in FIG. 1).

By “n−1” it is meant the next nucleotide position in the support strandrelative to the position which is occupied by, or has been occupied by,the universal nucleotide, in the direction distal to the helperstrand/proximal to the primer strand (nucleotide labelled “H” atposition n−1, as shown schematically in steps (1) and (2) of FIG. 1).n−1 may also refer to the corresponding position in the synthesisstrand. Thus at the cleavage step, position n−1 in the support strand isopposite the position occupied by the terminal nucleotide of the primerstrand portion of the synthesis strand (i.e. the nucleotide labelled “I”at position n−1, as shown schematically in step (2) of FIG. 1). Inmethods according to version 1, the nucleotide pair which occupypositions H and I can be any nucleotide pair.

Upon cleavage of the support strand between nucleotide positions n andn−1, the universal nucleotide, helper strand, if present immediatelyprior to cleavage, and the portion of the support strand which is or washybridized to the helper strand are removed from the remaining cleavedscaffold polynucleotide (see structure depicted at the top right of theupper part of FIG. 1).

A phosphate group should continue to be attached to the terminalnucleotide of the support strand of the cleaved scaffold polynucleotideat the cleavage site (as depicted in the structure shown in the middleof the lower part of FIG. 1). This ensures that the support strand ofthe ligation polynucleotide can be ligated to the support strand of thecleaved scaffold polynucleotide in the ligation step.

Thus in method version 1 the universal nucleotide occupies position n inthe support strand at steps (1) and (2) and the support strand iscleaved between nucleotide positions n and n−1.

Preferably, the support strand is cleaved by cleavage of thephosphodiester bond between nucleotide positions n and n−1 (the firstphosphodiester bond of the support strand relative to the position ofthe universal nucleotide, in the direction distal to the helperstrand/proximal to the primer strand).

The support strand may be cleaved by cleavage of one ester bond of thephosphodiester bond between nucleotide positions n and n−1.

Preferably the support strand is cleaved by cleavage of the first esterbond relative to nucleotide position n. This will have the effect ofretaining a terminal phosphate group on the support strand of thecleaved scaffold polynucleotide at the cleavage position.

Any suitable mechanism may be employed to effect cleavage of the supportstrand between nucleotide positions n and n−1 when the universalnucleotide occupies position n.

Cleavage of the support strand between nucleotide positions n and n−1 asdescribed above may be performed by the action of an enzyme.

Cleavage of the support strand between nucleotide positions n and n−1 asdescribed above may be performed as a two-step cleavage process.

The first cleavage step of a two-step cleavage process may compriseremoving the universal nucleotide from the support strand thus formingan abasic site at position n, and the second cleavage step may comprisecleaving the support strand at the abasic site, between positions n andn−1.

One mechanism of cleaving the support strand at a cleavage site definedby a sequence comprising a universal nucleotide which is occupyingposition n in the support strand is described in Example 2. The cleavagemechanism described in Example 2 is exemplary and other mechanisms couldbe employed, provided that the blunt-ended cleaved double-strandedscaffold polynucleotide described above is achieved.

In the first cleavage step of a two-step cleavage process the universalnucleotide is removed from the support strand whilst leaving thesugar-phosphate backbone intact. This can be achieved by the action ofan enzyme which can specifically excise a single universal nucleotidefrom a double-stranded polynucleotide. In the exemplified cleavagemethods the universal nucleotide is inosine and inosine is excised fromthe support strand by the action of an enzyme, thus forming an abasicsite. In the exemplified cleavage method the enzyme is a 3-methyladenineDNA glycosylase enzyme, specifically human alkyladenine DNA glycosylase(hAAG). Other enzymes, molecules or chemicals could be used providedthat an abasic site is formed. The nucleotide-excising enzyme may be UDGDNA glycosylase.

In the second cleavage step of a two-step cleavage process the supportstrand is cleaved at the abasic site by making a single-strand break. Inthe exemplified methods the support strand is cleaved by the action of achemical which is a base, such as NaOH. Alternatively, an organicchemical such as N,N′-dimethylethylenediamine may be used.Alternatively, enzymes having abasic site lyase activity, such as APEndonuclease 1, Endonuclease III (Nth), or Endonuclease VIII, may beused. Other enzymes, molecules or chemicals could be used provided thatthe support strand is cleaved at the abasic site as described.

Thus in embodiments wherein the universal nucleotide is at position n ofthe support strand at steps (1) and (2) and the support strand iscleaved between positions n and n−1, a first cleavage step may beperformed with a nucleotide-excising enzyme. An example of such anenzyme is a 3-methyladenine DNA glycosylase enzyme, such as humanalkyladenine DNA glycosylase (hAAG). The second cleavage step may beperformed with a chemical which is a base, such as NaOH. The second stepmay be performed with an organic chemical having abasic site cleavageactivity such as N,N′-dimethylethylenediamine. The second step mayperformed with an enzyme having abasic site lyase activity such asEndonuclease VIII.

Cleavage of the support strand between nucleotide positions n and n−1 asdescribed above may also be performed as a one-step cleavage process.Examples of enzymes which may be used in any such process includeEndonuclease III, Endonuclease VIII, formamidopirimidine DNA glycosylase(Fpg) and 8-oxoguanine DNA glycosylase (hOGG1).

In this exemplary method version of the invention, as well as with allversions, to allow the next nucleotide to be incorporated in the nextsynthesis cycle, the reversible terminator group must be removed fromthe first nucleotide (deprotection step; 104). This can be performed atvarious stages of the first cycle. Typically, and preferably, it will beperformed as step (4) of the method, before ligation step (5), as shownin step 4 of FIG. 1 (104). However, the deprotection step could beperformed at any step after incorporation of the new nucleotide, such asafter the ligation step (5). Regardless of which stage the deprotectionstep is performed, enzyme and residual unincorporated first nucleotidesshould first be removed in order to prevent multiple incorporation offirst nucleotides. Enzyme and unincorporated first nucleotides arepreferably removed prior to the ligation step (step (5)).

Removal of the reversible terminator group from the first nucleotide canbe performed by any suitable means. For example, removal can beperformed by the use of a chemical, such as tris(carboxyethyl)phosphine(TCEP).

In step (5) of the method a double-stranded ligation polynucleotide isligated (105) to the cleaved scaffold polynucleotide. The ligationpolynucleotide comprises a support strand and a helper strand. Theligation polynucleotide further comprises a complementary ligation endcomprising in the support strand a universal nucleotide and a singleoverhanging nucleotide which overhangs the terminal nucleotide of thehelper strand and which is the partner nucleotide for the firstnucleotide of the predefined sequence. The ligation polynucleotidefurther comprises in the helper strand adjacent the overhang a terminalnucleotide which is configured such that it cannot be ligated to anotherpolynucleotide strand (position labelled “X” in the structure depictedat the top left of the upper part of FIG. 1). Typically this terminalnucleotide will lack a phosphate group. Typically, as described above,this terminal nucleotide of the helper strand will define the 5′terminus of the helper strand.

The complementary ligation end is configured so that it will compatiblyjoin with the overhanging end of the cleaved scaffold polynucleotideproduct following incorporation step (3) when subjected to suitableligation conditions. Upon ligation of the support strands, the firstnucleotide becomes paired with its partner nucleotide.

Ligation of the support strands may be performed by any suitable means.Ligation will result in the joining of the support strands only, withthe maintenance of a single-stranded break between the first nucleotidein the synthesis strand, i.e. in the primer strand portion, and theterminal nucleotide of the helper strand adjacent the first nucleotide.

Ligation may typically be performed by enzymes having ligase activity.For example, ligation may be performed with T3 DNA ligase or T4 DNAligase or functional variants or equivalents thereof. The use of suchenzymes will result in the maintenance of the single-stranded break inthe synthesis strand, since the terminal nucleotide of the helper strandis provided such that it cannot act as a substrate for ligase, e.g. dueto the absence of a terminal phosphate group.

Ligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide completes a first synthesis cycle whereupon the scaffoldpolynucleotide of step (1) is effectively re-constituted except that thefirst nucleotide of the predefined nucleotide sequence is incorporatedinto the scaffold polynucleotide opposite its partner nucleotide.

In step (5) of this exemplary method (105), in the complementaryligation end of the ligation polynucleotide the universal nucleotide inthe support strand is positioned opposite the terminal nucleotide of thehelper strand and is paired therewith. It will be noted that in thecontext of the ligation polynucleotide the universal nucleotide ispositioned at position n+1, and at a position next to the terminalnucleotide of the support strand (position n). Position n in the contextof the ligation polynucleotide has the same meaning as described abovein the context of the scaffold polynucleotide at steps (1) and (2). Thusin step (5) by “n” it is meant the nucleotide position in the supportstrand which, following ligation, is opposite the position in thesynthesis strand which is now occupied by the nucleotide of thepredefined sequence following its addition to the terminal end of theprimer strand portion of the synthesis strand in that cycle ofsynthesis. By “n” it is also meant the nucleotide position in thesynthesis strand which, following ligation, is now occupied by thenucleotide of the predefined sequence following its addition to theterminal end of the primer strand portion of the synthesis strand inthat cycle of synthesis.

It will be noted that the universal nucleotide occupies position n inthe scaffold polynucleotide during steps (1) and (2) (101 and 102 ofFIG. 1), whereas the universal nucleotide occupies position n+1 in theligation polynucleotide in the same synthesis cycle (105 of FIG. 1).This is because at step (5) the cleaved double-stranded scaffoldpolynucleotide is now provided with the nucleotide of the predefinedsequence following its addition in that cycle of synthesis and becausethe ligation polynucleotide is provided with the nucleotide to partnerthe nucleotide of the predefined sequence; and furthermore because asdefined herein “n” always refers to the nucleotide position in thesynthesis strand which is occupied by (or will be occupied by) thenucleotide of the predefined sequence incorporated in that cycle orrefers to the nucleotide position opposite thereto in the supportstrand. Thus at the end of any given cycle of synthesis, immediatelyafter ligation step (5), the position occupied by a universal nucleotidewill be n+1 and will have moved from n, the position a universalnucleotide will have occupied at step (1). The product of the ligationstep (5) (structure labelled “ligation product” in FIG. 1) will be thescaffold polynucleotide for use in the next cycle of synthesis. When theligation product is considered as the scaffold polynucleotide for use inthe next cycle of synthesis (106) it is to be understood that theposition occupied by the universal nucleotide is now to be once againreferred to as position n (106), rather than n+1 (105), since theposition occupied by the universal nucleotide in the next cycle ofsynthesis (106) will be the nucleotide position in the support strandwhich is opposite the position in the synthesis strand which will beoccupied by the next nucleotide of the predefined sequence upon itsaddition to the terminal end of the cleaved synthesis strand in the nextcycle (108). Thus in method version 1 the position occupied by theuniversal nucleotide at the beginning of any given cycle of synthesis(101, 106 etc.) is always referred to as position n, and the nucleotidewhich is newly incorporated in that cycle always occupies a positionreferred to as n.

Following completion of the first synthesis cycle, second and subsequentcycles are performed using the same method steps.

As in step (2) of the first synthesis cycle of method version 1, in step(6) the scaffold polynucleotide is cleaved (107) at a cleavage site. Thecleavage site is defined by a sequence comprising the universalnucleotide in the support strand. Cleavage results in a double-strandedbreak in the scaffold polynucleotide. Cleavage of the scaffoldpolynucleotide leaves in place a cleaved double-stranded scaffoldpolynucleotide comprising, at the site of cleavage, a cleaved terminalend of the support strand and the terminal end of the primer strandportion of the synthesis strand which comprised the nick site prior tocleavage. Cleavage results in removal of the support strand comprisingthe universal nucleotide and the helper strand hybridised to the supportstrand, if present immediately prior to cleavage.

The cleavage steps may be performed as described above for step (2) ofthe first cycle.

In step (7) the next nucleotide is added to the terminal end of thecleaved primer strand portion of the synthesis strand as in step (3) ofthe first cycle.

In step (8) the reversible terminator group is removed from the nextnucleotide (deprotection step; 109). As described above for first cycle,this can be performed at various stages. Typically, and preferably, itwill be performed as step (8) of the method, before ligation step (9).However, the deprotection step could be performed at any step afterincorporation of the new nucleotide, such as after the ligation step(9).

Deprotection of the reversible terminator group in the next cycle (109)and subsequent cycles may be performed as described above with respectto the first synthesis cycle.

In step (9) of the next cycle a double-stranded ligation polynucleotideis ligated (110) to the cleaved scaffold polynucleotide. The ligationpolynucleotide of step (9) of the next and subsequent synthesis cyclesmay be configured, and the ligation step may be performed, as describedabove for step (5) of the first synthesis cycle.

Synthesis cycles are repeated as described above for as many times asnecessary to synthesise the double-stranded polynucleotide having thepredefined nucleotide sequence.

Synthesis Method Version 2

A second exemplary version of the synthesis method of the invention isperformed in an analogous matter to the first version except withmodified structural arrangements for the positioning of the universalnucleotide relative to the position to be occupied by the new nucleotideto be incorporated in a given synthesis cycle and relative to the nickand cleavage sites.

Thus as with version 1, in version 2 cleavage of the scaffoldpolynucleotide (step 2, 202) results in loss of the helper strand, ifpresent immediately prior to cleavage, and loss of the support strandcomprising the universal nucleotide. Cleavage leaves in place a cleaveddouble-stranded scaffold polynucleotide comprising, at the site ofcleavage, a cleaved terminal end of the support strand and the terminalend of the primer strand portion of the synthesis strand which comprisedthe nick site prior to cleavage. Cleavage results in a blunt-endedcleaved double-stranded scaffold polynucleotide at the site of cleavage,with no overhang.

In step (3) of the methods a first nucleotide in the predefinednucleotide sequence is added to the terminal end of the primer strandportion of the synthesis strand by the action of an enzyme havingtransferase activity such as polymerase or a terminal nucleotidyltransferase enzyme (203). The first nucleotide is provided with areversible terminator group which prevents further extension by theenzyme. Thus in step (3) only a single nucleotide is incorporated.

In step (4) of the methods a deprotection step is performed (204) toremove the terminator group from the newly-incorporated nucleotide. Inprinciple the deprotection step may be performed after the ligation step(5). Performance of the deprotection step as step (4) is preferred.

In step (5) of the methods a ligation polynucleotide (see structuredepicted at the top left of the upper part of FIG. 2) is ligated to thecleaved scaffold polynucleotide. Ligation incorporates a partnernucleotide into the scaffold polynucleotide and allows thenewly-incorporated nucleotide to pair with the partner nucleotide (step5 of FIG. 2; 205), thus completing a synthesis cycle.

In the second exemplary version of the synthesis method of the inventiona scaffold polynucleotide is provided in step (1) (201) as describedabove. In this method the universal nucleotide in the support strand ofthe scaffold polynucleotide is positioned opposite the penultimatenucleotide of the helper strand adjacent the single-strand break site(labelled “X” in the structures of FIG. 2), and is paired therewith (seestructure depicted in step 1 of FIG. 2).

In step (2) of the method the scaffold polynucleotide is cleaved (202)at a cleavage site. The cleavage site is defined by a sequencecomprising the universal nucleotide in the support strand. Cleavageresults in a double-stranded break in the scaffold polynucleotide.Cleavage of the scaffold polynucleotide (step 2) results in loss of thehelper strand, if present immediately prior to cleavage, and loss of thesupport strand comprising the universal nucleotide. Cleavage of thescaffold polynucleotide leaves in place a cleaved double-strandedscaffold polynucleotide comprising, at the site of cleavage, a cleavedterminal end of the support strand and the terminal end of the primerstrand portion of the synthesis strand which comprised the nick siteprior to cleavage.

The synthesis strand is already provided with a single-stranded break or“nick” in this exemplary method, thus only cleavage of the supportstrand is necessary to provide a double-stranded break in the scaffoldpolynucleotide.

In this exemplary method version cleavage generates a blunt-endedcleaved double-stranded scaffold polynucleotide with no overhang ineither the synthesis strand or the support strand.

In this method the universal nucleotide occupies position “n+1” in thesupport strand prior to the cleavage step. To obtain such a blunt-endedcleaved double-stranded scaffold polynucleotide when the universalnucleotide occupies position n+1 in the support strand, the supportstrand is cleaved at a specific position relative to the universalnucleotide. The support strand of the scaffold polynucleotide is cleavedbetween nucleotide positions n and n−1.

By “n” it is meant the nucleotide position in the support strand whichis opposite the position in the synthesis strand which will be occupiedby the nucleotide of the predefined sequence upon its addition to theterminal end of the cleaved synthesis strand in that cycle of synthesis.By “n” it is also meant the nucleotide position in the synthesis strandwhich will be occupied by the nucleotide of the predefined sequence uponits addition to the terminal end of the primer strand portion of thesynthesis strand in that cycle of synthesis. Thus at the cleavage step,the universal nucleotide is at position n+1 in the support strand, whichis opposite the position occupied by the penultimate nucleotide of thehelper strand adjacent the nick site (shown at position “X” in FIG. 2).

At steps (1) and (2) the position which will be occupied by thenucleotide of the predefined sequence incorporated in that cycle ofsynthesis corresponds with the terminal nucleotide of the helper strandportion of the synthesis strand (shown at position “I” in FIG. 2).

By “n+1” it is meant the next nucleotide position in the support strandrelative to position n in the direction proximal to the helperstrand/distal to the primer strand (nucleotide labelled “Un” at positionn+1, which occupies the position next to the position labelled “H” atposition n in the direction distal to the primer strand, as shownschematically in steps (1) and (2) of FIG. 2). n+1 may also refer to thecorresponding position in the synthesis strand (labelled “X” at positionn+1 in steps (1) and (2) of FIG. 2). Thus at the cleavage step, positionn+1 in the support strand is opposite the position occupied by thepenultimate nucleotide of the helper strand adjacent the nick site.

By “n−1” it is meant the next nucleotide position in the support strandrelative to position n in the direction distal to the helperstrand/proximal to the primer strand (position labelled “J” at positionn−1, as shown schematically in steps (1) and (2) of FIG. 2). n−1 mayalso refer to the corresponding position in the synthesis strand (i.e.the position labelled “K” at position n−1, as shown schematically instep (2) of FIG. 2). Thus at the cleavage step, position n−1 in thesupport strand is opposite the position occupied by the terminalnucleotide of the primer strand portion of the synthesis strand.

In methods according to version 2, the nucleotide pair which occupypositions H and I, and J and K, can be any nucleotide pair.

Upon cleavage of the support strand between nucleotide positions n andn−1, the universal nucleotide, helper strand (if present) and theportion of the support strand which is or was hybridized to the helperstrand are removed from the remaining cleaved scaffold polynucleotide(see structure depicted at the top right of the upper part of FIG. 2).

A phosphate group should continue to be attached to the terminalnucleotide of the support strand of the cleaved scaffold polynucleotideat the cleavage site (as depicted in the structure shown in the middleof the lower part of FIG. 2). This ensures that the support strand ofthe ligation polynucleotide can be ligated to the support strand of thecleaved scaffold polynucleotide in the ligation step.

Thus in method version 2 the universal nucleotide occupies position n+1in the support strand at steps (1) and (2) and the support strand iscleaved between nucleotide positions n and n−1.

Preferably, the support strand is cleaved by cleavage of thephosphodiester bond between nucleotide positions n and n−1 (the secondphosphodiester bond of the support strand relative to the position ofthe universal nucleotide, in the direction distal to the helperstrand/proximal to the primer strand).

The support strand may be cleaved by cleavage of one ester bond of thephosphodiester bond between nucleotide positions n and n−1.

Preferably the support strand is cleaved by cleavage of the first esterbond relative to nucleotide position n. This will have the effect ofretaining a terminal phosphate group on the support strand of thecleaved scaffold polynucleotide at the cleavage position.

Any suitable mechanism may be employed to effect cleavage of the supportstrand between nucleotide positions n and n−1 when the universalnucleotide occupies position n+1.

Cleavage of the support strand between nucleotide positions n and n−1 asdescribed above may be performed by the action of an enzyme.

Cleavage of the support strand between nucleotide positions n and n−1when the universal nucleotide occupies position n+1 in the supportstrand, as described above, may be performed by the action of an enzymesuch as Endonuclease V.

One mechanism of cleaving the support strand between nucleotidepositions n and n−1 at a cleavage site defined by a sequence comprisinga universal nucleotide which is occupying position n+1 in the supportstrand is described in analogous fashion in Example 3. The mechanismdescribed is exemplary and other mechanisms could be employed, providedthat the cleavage arrangement described above is achieved.

In this exemplary mechanism an endonuclease enzyme is employed. In theexemplified method the enzyme is Endonuclease V. Other enzymes,molecules or chemicals could be used provided that the support strand iscleaved between nucleotide positions n and n−1 when the universalnucleotide occupies position n+1 in the support strand.

In this exemplary method version of the invention, as well as with allversions, to allow the next nucleotide to be incorporated in the nextsynthesis cycle, the reversible terminator group must be removed fromthe first nucleotide (deprotection step; 204). This can be performed atvarious stages of the first cycle. Typically, and preferably, it will beperformed as step (4) of the method, before ligation step (5), as shownin step 4 of FIG. 2 (204). However, the deprotection step could beperformed at any step after incorporation of the new nucleotide, such asafter the ligation step (5). Regardless of which stage the deprotectionstep is performed, enzyme and residual unincorporated first nucleotidesshould first be removed in order to prevent multiple incorporation offirst nucleotides. Enzyme and unincorporated first nucleotides arepreferably removed prior to the ligation step (step (5)).

Removal of the reversible terminator group from the first nucleotide canbe performed by any suitable means. For example, removal can beperformed by the use of a chemical, such as tris(carboxyethyl)phosphine(TCEP).

In step (5) of the method a double-stranded ligation polynucleotide isligated (205) to the cleaved scaffold polynucleotide. The ligationpolynucleotide comprises a support strand and a helper strand. Theligation polynucleotide further comprises a complementary ligation endcomprising in the support strand a universal nucleotide and a singleoverhanging nucleotide which overhangs the terminal nucleotide of thehelper strand and which is the partner nucleotide for the firstnucleotide of the predefined sequence. The ligation polynucleotidefurther comprises in the helper strand adjacent the overhang a terminalnucleotide which is configured such that it cannot be ligated to anotherpolynucleotide strand (position labelled “I” in the structure depictedat the top left of the upper part of FIG. 2). Typically this terminalnucleotide will lack a phosphate group. Typically, as described above,this terminal nucleotide of the helper strand will define the 5′terminus of the helper strand.

In the complementary ligation end of the ligation polynucleotide theuniversal nucleotide in the support strand is positioned opposite thepenultimate nucleotide of the helper strand (position labelled “X” instep (5) of FIG. 2) and is paired therewith. The terminal nucleotide ofthe helper strand (position labelled “I” in step (5) of FIG. 2 atposition n+1) is paired with a nucleotide in the support strand whichoccupies a position (position labelled “H” in step (5) of FIG. 2 atposition n+1) which is between the universal nucleotide at position n+2and the partner nucleotide at position n.

The complementary ligation end is configured so that it will compatiblyjoin with the overhanging end of the cleaved scaffold polynucleotideproduct following incorporation step (3) when subjected to suitableligation conditions. Upon ligation of the support strands, the firstnucleotide becomes paired with its partner nucleotide.

Ligation of the support strands may be performed by any suitable means.Ligation will result in the joining of the support strands only, withthe maintenance of a single-stranded break between the first nucleotidein the synthesis strand, i.e. in the primer strand portion, and theterminal nucleotide of the helper strand adjacent the first nucleotide.

Ligation may typically be performed by enzymes having ligase activity.For example, ligation may be performed with T3 DNA ligase or T4 DNAligase or functional variants or equivalents thereof. The use of suchenzymes will result in the maintenance of the single-stranded break inthe synthesis strand, since the terminal nucleotide of the helper strandis provided such that it cannot act as a substrate for ligase, e.g. dueto the absence of a terminal phosphate group.

Ligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide completes a first synthesis cycle whereupon the scaffoldpolynucleotide of step (1) is effectively re-constituted except that thefirst nucleotide of the predefined nucleotide sequence is incorporatedinto the scaffold polynucleotide opposite its partner nucleotide.

In step (5) of this exemplary method (205), in the complementaryligation end of the ligation polynucleotide the universal nucleotide inthe support strand is positioned opposite the penultimate nucleotide ofthe helper strand (position labelled “X”) and is paired therewith. Itwill be noted that in the context of the ligation polynucleotide theuniversal nucleotide is positioned at position n+2, i.e. two positionsremoved from the terminal nucleotide of the support strand (position n).Position n in the context of the ligation polynucleotide has the samemeaning as described above in the context of the scaffold polynucleotideat steps (1) and (2). Thus in step (5) by “n” it is meant the nucleotideposition in the support strand which, following ligation, is oppositethe position in the synthesis strand which is now occupied by thenucleotide of the predefined sequence following its addition to theterminal end of the primer strand portion of the synthesis strand inthat cycle of synthesis. By “n” it is also meant the nucleotide positionin the synthesis strand following ligation which is now occupied by thenucleotide of the predefined sequence following its addition to theterminal end of the primer strand portion of the synthesis strand inthat cycle of synthesis.

It will be noted that the universal nucleotide occupies position n+1 inthe scaffold polynucleotide during steps (1) and (2) (201 and 202 ofFIG. 2), whereas the universal nucleotide occupies position n+2 in theligation polynucleotide in the same synthesis cycle (205 of FIG. 2).This is because at step (5) the cleaved double-stranded scaffoldpolynucleotide is now provided with the nucleotide of the predefinedsequence following its addition in that cycle of synthesis and becausethe ligation polynucleotide is provided with the nucleotide to partnerthe nucleotide of the predefined sequence; and furthermore because asdefined herein “n” always refers to the nucleotide position in thesynthesis strand which is occupied by (or will be occupied by) thenucleotide of the predefined sequence incorporated in that cycle orrefers to the nucleotide position opposite thereto in the supportstrand. Thus at the end of any given cycle of synthesis, immediatelyafter ligation step (5), the position occupied by a universal nucleotidewill be n+2 and will have moved from n+1, the position a universalnucleotide will have occupied at step (1). The product of the ligationstep (5) (structure labelled “ligation product” in FIG. 2) will be thescaffold polynucleotide for use in the next cycle of synthesis. When theligation product is considered as the scaffold polynucleotide for use inthe next cycle of synthesis (206) it is to be understood that theposition occupied by the universal nucleotide is now to be once againreferred to as position n+1 (206), rather than n+2 (205). Thus in methodversion 2 the position occupied by the universal nucleotide at thebeginning of any given cycle of synthesis (201, 206 etc.) is alwaysreferred to as position n+1, and the nucleotide which is newlyincorporated in that cycle always occupies a position referred to as n.

Following completion of the first synthesis cycle, second and subsequentcycles are performed using the same method steps.

As in step (2) of the first synthesis cycle of method version 2, in step(6) the scaffold polynucleotide is cleaved (207) at a cleavage site. Thecleavage site is defined by a sequence comprising the universalnucleotide in the support strand. Cleavage results in a double-strandedbreak in the scaffold polynucleotide. Cleavage of the scaffoldpolynucleotide leaves in place a cleaved double-stranded scaffoldpolynucleotide comprising, at the site of cleavage, a cleaved terminalend of the support strand and the terminal end of the primer strandportion of the synthesis strand which comprised the nick site prior tocleavage. Cleavage results in removal of the support strand comprisingthe universal nucleotide and the helper strand hybridised to the supportstrand, if present immediately prior to cleavage.

The cleavage steps may be performed as described above for step (2) ofthe first cycle.

In step (7) the next nucleotide is added to the terminal end of thecleaved primer strand portion of the synthesis strand as in step (3) ofthe first cycle.

In step (8) the reversible terminator group is removed from the nextnucleotide (deprotection step; 209). As described above for first cycle,this can be performed at various stages. Typically, and preferably, itwill be performed as step (8) of the method, before ligation step (9).However, the deprotection step could be performed at any step afterincorporation of the new nucleotide, such as after the ligation step(9).

Deprotection of the reversible terminator group in the next cycle (209)and subsequent cycles may be performed as described above with respectto the first synthesis cycle.

In step (9) of the next cycle a double-stranded ligation polynucleotideis ligated (210) to the cleaved scaffold polynucleotide. The ligationpolynucleotide of step (9) of the next and subsequent synthesis cyclesmay be configured, and the ligation step may be performed, as describedabove for step (5) of the first synthesis cycle.

Synthesis cycles are repeated as described above for as many times asnecessary to synthesise the double-stranded polynucleotide having thepredefined nucleotide sequence.

Synthesis Method Version 3

A third exemplary version of the synthesis method of the invention isperformed in an analogous matter to the first version except withmodified structural arrangements for the positioning of the universalnucleotide relative to the cleavage site.

Thus as with version 1, in version 3 cleavage of the scaffoldpolynucleotide (step 2, 302) results in loss of the helper strand, ifpresent immediately prior to cleavage, and loss of the support strandcomprising the universal nucleotide. Cleavage leaves in place a cleaveddouble-stranded scaffold polynucleotide comprising, at the site ofcleavage, a cleaved terminal end of the support strand and the terminalend of the primer strand portion of the synthesis strand which comprisedthe nick site prior to cleavage. Cleavage results in a cleaveddouble-stranded scaffold polynucleotide with a single nucleotideoverhang at the site of cleavage.

In step (3) of the methods a first nucleotide in the predefinednucleotide sequence is added to the terminal end of the primer strandportion of the synthesis strand by the action of an enzyme havingtransferase activity such as polymerase or a terminal nucleotidyltransferase enzyme (303). The first nucleotide is provided with areversible terminator group which prevents further extension by theenzyme. Thus in step (3) only a single nucleotide is incorporated.

In step (4) of the methods a deprotection step is performed (304) toremove the terminator group from the newly-incorporated nucleotide. Inprinciple the deprotection step may be performed after the ligation step(5). Performance of the deprotection step as step (4) is preferred.

In step (5) of the methods a ligation polynucleotide (see structuredepicted at the top left of the upper part of FIG. 3) is ligated to thecleaved scaffold polynucleotide. Ligation incorporates a partnernucleotide into the scaffold polynucleotide and allows thenewly-incorporated nucleotide to pair with the partner nucleotide (step5 of FIG. 3; 305), thus completing a synthesis cycle.

In the third exemplary version of the synthesis method of the inventiona scaffold polynucleotide is provided in step (1) (301) as describedabove. In this method the universal nucleotide in the support strand ofthe scaffold polynucleotide is positioned opposite the terminalnucleotide of the helper strand adjacent the single-strand break site(labelled “X” in the structures of FIG. 3), and is paired therewith (seestructure depicted in step 1 of FIG. 3).

In step (2) of the method the scaffold polynucleotide is cleaved (302)at a cleavage site. The cleavage site is defined by a sequencecomprising the universal nucleotide in the support strand. Cleavageresults in a double-stranded break in the scaffold polynucleotide.Cleavage of the scaffold polynucleotide (step 2) results in loss of thehelper strand, if present immediately prior to cleavage, and loss of thesupport strand comprising the universal nucleotide. Cleavage of thescaffold polynucleotide leaves in place a cleaved double-strandedscaffold polynucleotide comprising, at the site of cleavage, a cleavedterminal end of the support strand and the terminal end of the primerstrand portion of the synthesis strand which comprised the nick siteprior to cleavage.

The synthesis strand is already provided with a single-stranded break or“nick” in this exemplary method, thus only cleavage of the supportstrand is necessary to provide a double-stranded break in the scaffoldpolynucleotide.

In this exemplary method version cleavage generates a cleaveddouble-stranded scaffold polynucleotide with a single nucleotideoverhang. Thus the terminal nucleotide of the primer strand portion ofthe synthesis strand overhangs the terminal nucleotide of the supportstrand.

In this method the universal nucleotide occupies position “n” in thesupport strand prior to the cleavage step. To obtain such a cleaveddouble-stranded scaffold polynucleotide wherein the terminal nucleotideof the primer strand portion of the synthesis strand overhangs theterminal nucleotide of the support strand when the universal nucleotideoccupies position n in the support strand, the support strand is cleavedat a specific position relative to the universal nucleotide and relativeto the nick site. The support strand of the scaffold polynucleotide iscleaved between nucleotide positions n−1 and n−2.

By “n” it is meant the nucleotide position in the support strand whichis opposite the position in the synthesis strand which will be occupiedby the nucleotide of the predefined sequence upon its addition to theterminal end of the cleaved synthesis strand in that cycle of synthesis.By “n” it is also meant the nucleotide position in the synthesis strandwhich will be occupied by the nucleotide of the predefined sequence uponits addition to the terminal end of the primer strand portion of thesynthesis strand in that cycle of synthesis. Thus at the cleavage step,the universal nucleotide is at position n in the support strand, whichis opposite the position occupied by the terminal nucleotide of thehelper strand adjacent the nick site (shown at position “X” in FIG. 3).

At steps (1) and (2) the position which will be occupied by thenucleotide of the predefined sequence incorporated in that cycle ofsynthesis corresponds with the terminal nucleotide of the helper strandportion of the synthesis strand (shown at position “X” in FIG. 3).

By “n−1” it is meant the next nucleotide position in the support strandrelative to position n in the direction distal to the helperstrand/proximal to the primer strand (position labelled “H” at positionn−1, as shown schematically in steps (1) and (2) of FIG. 3). n−1 mayalso refer to the corresponding position in the synthesis strand (i.e.the position labelled “I” at position n−1, as shown schematically instep (2) of FIG. 3). Thus at the cleavage step, position n−1 in thesupport strand is opposite the position occupied by the terminalnucleotide of the primer strand portion of the synthesis strand. Insteps (1) and (2) of FIG. 3 the nucleotides at position “J” and “K” thusoccupy position n−2.

In methods according to version 3, the nucleotide pair which occupypositions H and I, and J and K, can be any nucleotide pair.

Upon cleavage of the support strand between nucleotide positions n−1 andn−2, the universal nucleotide, helper strand (if present) and theportion of the support strand which is or was hybridized to the helperstrand are removed from the remaining cleaved scaffold polynucleotide(see structure depicted at the top right of the upper part of FIG. 3).

A phosphate group should continue to be attached to the terminalnucleotide of the support strand of the cleaved scaffold polynucleotideat the cleavage site (as depicted in the structure shown in the middleof the lower part of FIG. 3). This ensures that the support strand ofthe ligation polynucleotide can be ligated to the support strand of thecleaved scaffold polynucleotide in the ligation step.

Thus in method version 3 the universal nucleotide occupies position n inthe support strand at steps (1) and (2) and the support strand iscleaved between nucleotide positions n−1 and n−2.

Preferably, the support strand is cleaved by cleavage of thephosphodiester bond between nucleotide positions n−1 and n−2 (the secondphosphodiester bond of the support strand relative to the position ofthe universal nucleotide, in the direction distal to the helperstrand/proximal to the primer strand).

The support strand may be cleaved by cleavage of one ester bond of thephosphodiester bond between nucleotide positions n−1 and n−2.

Preferably the support strand is cleaved by cleavage of the first esterbond relative to nucleotide position n−1. This will have the effect ofretaining a terminal phosphate group on the support strand of thecleaved scaffold polynucleotide at the cleavage position.

Any suitable mechanism may be employed to effect cleavage of the supportstrand between nucleotide positions n−1 and n−2 when the universalnucleotide occupies position n.

Cleavage of the support strand between nucleotide positions n−1 and n−2as described above may be performed by the action of an enzyme.

Cleavage of the support strand between nucleotide positions n−1 and n−2when the universal nucleotide occupies position n in the support strand,as described above, may be performed by the action of an enzyme such asEndonuclease V.

One mechanism of cleaving the support strand between nucleotidepositions n−1 and n−2 at a cleavage site defined by a sequencecomprising a universal nucleotide which is occupying position n in thesupport strand is described in analogous fashion in Example 3. Themechanism described is exemplary and other mechanisms could be employed,provided that the cleavage arrangement and single-nucleotide overhangdescribed above is achieved.

In this exemplary mechanism an endonuclease enzyme is employed. In theexemplified method the enzyme is Endonuclease V. Other enzymes,molecules or chemicals could be used provided that the support strand iscleaved between nucleotide positions n−1 and n−2 when the universalnucleotide occupies position n in the support strand.

In this exemplary method version of the invention, as well as with allversions, to allow the next nucleotide to be incorporated in the nextsynthesis cycle, the reversible terminator group must be removed fromthe first nucleotide (deprotection step; 304). This can be performed atvarious stages of the first cycle. Typically, and preferably, it will beperformed as step (4) of the method, before ligation step (5), as shownin step 4 of FIG. 3 (304). However, the deprotection step could beperformed at any step after incorporation of the new nucleotide, such asafter the ligation step (5). Regardless of which stage the deprotectionstep is performed, enzyme and residual unincorporated first nucleotidesshould first be removed in order to prevent multiple incorporation offirst nucleotides. Enzyme and unincorporated first nucleotides arepreferably removed prior to the ligation step (step (5)).

Removal of the reversible terminator group from the first nucleotide canbe performed by any suitable means. For example, removal can beperformed by the use of a chemical, such as tris(carboxyethyl)phosphine(TCEP).

In step (5) of the method a double-stranded ligation polynucleotide isligated (305) to the cleaved scaffold polynucleotide. The ligationpolynucleotide comprises a support strand and a helper strand. Theligation polynucleotide further comprises a complementary ligation endcomprising in the support strand a universal nucleotide and twooverhanging nucleotides which overhang the terminal nucleotide of thehelper strand. The penultimate nucleotide of the support strand of theligation polynucleotide at the complementary ligation end is the partnernucleotide for the first nucleotide of the predefined sequence. Theligation polynucleotide further comprises in the helper strand adjacentthe overhang a terminal nucleotide which is configured such that itcannot be ligated to another polynucleotide strand (position labelled“X” in the structure depicted at the top left of the upper part of FIG.3). Typically this terminal nucleotide will lack a phosphate group.Typically, as described above, this terminal nucleotide of the helperstrand will define the 5′ terminus of the helper strand.

In the complementary ligation end of the ligation polynucleotide theuniversal nucleotide in the support strand is positioned (n+1) oppositethe terminal nucleotide of the helper strand (position labelled “X” instep (5) of FIG. 3) and is paired therewith. The partner nucleotide forthe first nucleotide of the predefined sequence occupies the nextnucleotide position relative to the universal nucleotide in thedirection distal to the helper strand (position n) and is positionedbetween the universal nucleotide and the terminal nucleotide of thesupport strand of the ligation polynucleotide (position labelled “H” instep (5) of FIG. 3 at position n−1).

The complementary ligation end is configured so that it will compatiblyjoin with the overhanging end of the cleaved scaffold polynucleotideproduct following incorporation step (3) when subjected to suitableligation conditions. Upon ligation of the support strands, the firstnucleotide becomes paired with its partner nucleotide.

Ligation of the support strands may be performed by any suitable means.Ligation will result in the joining of the support strands only, withthe maintenance of a single-stranded break between the first nucleotidein the synthesis strand, i.e. in the primer strand portion, and theterminal nucleotide of the helper strand adjacent the first nucleotide.

Ligation may typically be performed by enzymes having ligase activity.For example, ligation may be performed with T3 DNA ligase or T4 DNAligase or functional variants or equivalents thereof. The use of suchenzymes will result in the maintenance of the single-stranded break inthe synthesis strand, since the terminal nucleotide of the helper strandis provided such that it cannot act as a substrate for ligase, e.g. dueto the absence of a terminal phosphate group.

Ligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide completes a first synthesis cycle whereupon the scaffoldpolynucleotide of step (1) is effectively re-constituted except that thefirst nucleotide of the predefined nucleotide sequence is incorporatedinto the scaffold polynucleotide opposite its partner nucleotide.

In step (5) of this exemplary method (305), in the complementaryligation end of the ligation polynucleotide the universal nucleotide inthe support strand is positioned opposite the terminal nucleotide of thehelper strand (position labelled “X”) and is paired therewith. It willbe noted that in the context of the ligation polynucleotide theuniversal nucleotide is positioned at position n+1 and two positionsremoved from the terminal nucleotide of the support strand (positionn−1). Position n in the context of the ligation polynucleotide has thesame meaning as described above in the context of the scaffoldpolynucleotide at steps (1) and (2). Thus in step (5) by “n” it is meantthe nucleotide position in the support strand which, following ligation,is opposite the position in the synthesis strand which is now occupiedby the nucleotide of the predefined sequence following its addition tothe terminal end of the primer strand portion of the synthesis strand inthat cycle of synthesis. By “n” it is also meant the nucleotide positionin the synthesis strand following ligation which is now occupied by thenucleotide of the predefined sequence following its addition to theterminal end of the primer strand portion of the synthesis strand inthat cycle of synthesis.

It will be noted that the universal nucleotide occupies position n inthe scaffold polynucleotide during steps (1) and (2) (301 and 302 ofFIG. 3), whereas the universal nucleotide occupies position n+1 in theligation polynucleotide in the same synthesis cycle (305 of FIG. 3).This is because at step (5) the cleaved double-stranded scaffoldpolynucleotide is now provided with the nucleotide of the predefinedsequence following its addition in that cycle of synthesis and becausethe ligation polynucleotide is provided with the nucleotide to partnerthe nucleotide of the predefined sequence; and furthermore because asdefined herein “n” always refers to the nucleotide position in thesynthesis strand which is occupied by (or will be occupied by) thenucleotide of the predefined sequence incorporated in that cycle orrefers to the nucleotide position opposite thereto in the supportstrand. Thus at the end of any given cycle of synthesis, immediatelyafter ligation step (5), the position occupied by a universal nucleotidewill be n+1 and will have moved from n, the position a universalnucleotide will have occupied at step (1). The product of the ligationstep (5) (structure labelled “ligation product” in FIG. 3) will be thescaffold polynucleotide for use in the next cycle of synthesis. When theligation product is considered as the scaffold polynucleotide for use inthe next cycle of synthesis (306) it is to be understood that theposition occupied by the universal nucleotide is now to be once againreferred to as position n (306), rather than n+1 (305). Thus in methodversion 3 the position occupied by the universal nucleotide at thebeginning of any given cycle of synthesis (301, 306 etc.) is alwaysreferred to as position n, and the nucleotide which is newlyincorporated in that cycle always occupies a position referred to as n.

Following completion of the first synthesis cycle, second and subsequentcycles are performed using the same method steps.

As in step (2) of the first synthesis cycle of method version 3, in step(6) the scaffold polynucleotide is cleaved (307) at a cleavage site. Thecleavage site is defined by a sequence comprising the universalnucleotide in the support strand. Cleavage results in a double-strandedbreak in the scaffold polynucleotide. Cleavage of the scaffoldpolynucleotide leaves in place a cleaved double-stranded scaffoldpolynucleotide comprising, at the site of cleavage, a cleaved terminalend of the support strand and the terminal end of the primer strandportion of the synthesis strand which comprised the nick site prior tocleavage. Cleavage results in removal of the support strand comprisingthe universal nucleotide and the helper strand hybridised to the supportstrand, if present immediately prior to cleavage.

The cleavage steps may be performed as described above for step (2) ofthe first cycle.

In step (7) the next nucleotide is added to the terminal end of thecleaved primer strand portion of the synthesis strand as in step (3) ofthe first cycle.

In step (8) the reversible terminator group is removed from the nextnucleotide (deprotection step; 309). As described above for first cycle,this can be performed at various stages. Typically, and preferably, itwill be performed as step (8) of the method, before ligation step (9).However, the deprotection step could be performed at any step afterincorporation of the new nucleotide, such as after the ligation step(9).

Deprotection of the reversible terminator group in the next cycle (309)and subsequent cycles may be performed as described above with respectto the first synthesis cycle.

In step (9) of the next cycle a double-stranded ligation polynucleotideis ligated (310) to the cleaved scaffold polynucleotide. The ligationpolynucleotide of step (9) of the next and subsequent synthesis cyclesmay be configured, and the ligation step may be performed, as describedabove for step (5) of the first synthesis cycle.

Synthesis cycles are repeated as described above for as many times asnecessary to synthesise the double-stranded polynucleotide having thepredefined nucleotide sequence.

Synthesis Method Version 4

A fourth exemplary version of the synthesis method of the invention isperformed in an analogous matter to the first version except withmodified structural arrangements for the positioning of the universalnucleotide relative to the position to be occupied by the new nucleotideto be incorporated in a given synthesis cycle and relative to the nickand cleavage sites.

Thus as with version 1, in version 4 cleavage of the scaffoldpolynucleotide (step 2, 402) results in loss of the helper strand, ifpresent immediately prior to cleavage, and loss of the support strandcomprising the universal nucleotide. Cleavage leaves in place a cleaveddouble-stranded scaffold polynucleotide comprising, at the site ofcleavage, a cleaved terminal end of the support strand and the terminalend of the primer strand portion of the synthesis strand which comprisedthe nick site prior to cleavage. Cleavage results in a blunt-endedcleaved double-stranded scaffold polynucleotide at the site of cleavage,with no overhang.

In step (3) of the methods a first nucleotide in the predefinednucleotide sequence is added to the terminal end of the primer strandportion of the synthesis strand by the action of an enzyme havingtransferase activity such as polymerase or a terminal nucleotidyltransferase enzyme (403). The first nucleotide is provided with areversible terminator group which prevents further extension by theenzyme. Thus in step (3) only a single nucleotide is incorporated.

In step (4) of the methods a deprotection step is performed (404) toremove the terminator group from the newly-incorporated nucleotide. Inprinciple the deprotection step may be performed after the ligation step(5). Performance of the deprotection step as step (4) is preferred.

In step (5) of the methods a ligation polynucleotide (see structuredepicted at the top left of the upper part of FIG. 4) is ligated to thecleaved scaffold polynucleotide. Ligation incorporates a partnernucleotide into the scaffold polynucleotide and allows thenewly-incorporated nucleotide to pair with the partner nucleotide (step5 of FIG. 4; 405), thus completing a synthesis cycle.

In the fourth exemplary version of the synthesis method of the inventiona scaffold polynucleotide is provided in step (1) (401) as describedabove. In this method the universal nucleotide in the support strand ofthe scaffold polynucleotide is positioned opposite and paired with anucleotide (labelled “X” in the structures of FIG. 4) which is twopositions removed from the terminal nucleotide of the helper strand(labelled “K” in the structure depicted in step (1) of FIG. 4) adjacentthe single-strand break site.

In step (2) of the method the scaffold polynucleotide is cleaved (402)at a cleavage site. The cleavage site is defined by a sequencecomprising the universal nucleotide in the support strand. Cleavageresults in a double-stranded break in the scaffold polynucleotide.Cleavage of the scaffold polynucleotide (step 2) results in loss of thehelper strand, if present immediately prior to cleavage, and loss of thesupport strand comprising the universal nucleotide. Cleavage of thescaffold polynucleotide leaves in place a cleaved double-strandedscaffold polynucleotide comprising, at the site of cleavage, a cleavedterminal end of the support strand and the terminal end of the primerstrand portion of the synthesis strand which comprised the nick siteprior to cleavage.

The synthesis strand is already provided with a single-stranded break or“nick” in this exemplary method, thus only cleavage of the supportstrand is necessary to provide a double-stranded break in the scaffoldpolynucleotide.

In this exemplary method version cleavage generates a blunt-endedcleaved double-stranded scaffold polynucleotide with no overhang.

In this method the universal nucleotide occupies position “n+2” in thesupport strand prior to the cleavage step. To obtain such a blunt-endedcleaved double-stranded scaffold polynucleotide when the universalnucleotide occupies position n+2 in the support strand, the supportstrand is cleaved at a specific position relative to the universalnucleotide and relative to the nick site. The support strand of thescaffold polynucleotide is cleaved between nucleotide positions n andn−1.

By “n” it is meant the nucleotide position in the support strand whichis opposite the position in the synthesis strand which will be occupiedby the nucleotide of the predefined sequence upon its addition to theterminal end of the cleaved synthesis strand in that cycle of synthesis.By “n” it is also meant the nucleotide position in the synthesis strandwhich will be occupied by the nucleotide of the predefined sequence uponits addition to the terminal end of the primer strand portion of thesynthesis strand in that cycle of synthesis. Thus at the cleavage step,the universal nucleotide is at position n+2 in the support strand, whichis opposite the nucleotide which is two positions removed from theterminal nucleotide of the helper strand adjacent the single-strandbreak site (shown at position “X” in step (2) in FIG. 4).

At steps (1) and (2) the position which will be occupied by thenucleotide of the predefined sequence incorporated in that cycle ofsynthesis corresponds with the nucleotide which is two positions removedfrom the terminal nucleotide of the helper strand adjacent thesingle-strand break site (shown at position “X” in step (2) of FIG. 4).

By “n+1” it is meant the next nucleotide position in the support strandrelative to position n in the direction proximal to the helperstrand/distal to the primer strand (nucleotide labelled “H” at positionn+1, which occupies the position next to the position labelled “J” atposition n in the direction distal to the primer strand, as shownschematically in steps (1) and (2) of FIG. 4). n+1 may also refer to thecorresponding position in the synthesis strand (labelled “I” at positionn+1 in steps (1) and (2) of FIG. 4). Thus at the cleavage step, positionn+1 in the support strand is opposite the position occupied by thepenultimate nucleotide of the helper strand adjacent the nick site.

By “n−1” it is meant the next nucleotide position in the support strandrelative to position n in the direction distal to the helperstrand/proximal to the primer strand (position labelled “L” at positionn−1, as shown schematically in steps (1) and (2) of FIG. 4). n−1 mayalso refer to the corresponding position in the synthesis strand (i.e.the position labelled “M” at position n−1, as shown schematically insteps (1) and (2) of FIG. 4). Thus at the cleavage step, position n−1 inthe support strand is opposite the position occupied by the terminalnucleotide of the primer strand portion of the synthesis strand. Insteps (1) and (2) of FIG. 4 the nucleotides at position “J” and “K” thusoccupy position n.

In methods according to version 4, the nucleotide pair which occupypositions H and I; J and K; and L and M can be any nucleotide pair.

Upon cleavage of the support strand between nucleotide positions n andn−1, the universal nucleotide, helper strand (if present) and theportion of the support strand which is or was hybridized to the helperstrand are removed from the remaining cleaved scaffold polynucleotide(see structure depicted at the top right of the upper part of FIG. 4).

A phosphate group should continue to be attached to the terminalnucleotide of the support strand of the cleaved scaffold polynucleotideat the cleavage site (as depicted in the structure shown in the middleof the lower part of FIG. 4). This ensures that the support strand ofthe ligation polynucleotide can be ligated to the support strand of thecleaved scaffold polynucleotide in the ligation step.

Thus in method version 4 the universal nucleotide occupies position n+2in the support strand at steps (1) and (2) and the support strand iscleaved between nucleotide positions n and n−1.

Preferably, the support strand is cleaved by cleavage of thephosphodiester bond between nucleotide positions n and n−1 (the thirdphosphodiester bond of the support strand relative to the position ofthe universal nucleotide, in the direction distal to the helperstrand/proximal to the primer strand).

The support strand may be cleaved by cleavage of one ester bond of thephosphodiester bond between nucleotide positions n and n−1.

Preferably the support strand is cleaved by cleavage of the first esterbond relative to nucleotide position n. This will have the effect ofretaining a terminal phosphate group on the support strand of thecleaved scaffold polynucleotide at the cleavage position.

Any suitable mechanism may be employed to effect cleavage of the supportstrand between nucleotide positions n and n−1 when the universalnucleotide occupies position n+2.

Cleavage of the support strand between nucleotide positions n and n−1 asdescribed above may be performed by the action of an enzyme.

In this exemplary method version of the invention, as well as with allversions, to allow the next nucleotide to be incorporated in the nextsynthesis cycle, the reversible terminator group must be removed fromthe first nucleotide (deprotection step; 404). This can be performed atvarious stages of the first cycle. Typically, and preferably, it will beperformed as step (4) of the method, before ligation step (5), as shownin step 4 of FIG. 4 (404). However, the deprotection step could beperformed at any step after incorporation of the new nucleotide, such asafter the ligation step (5). Regardless of which stage the deprotectionstep is performed, enzyme and residual unincorporated first nucleotidesshould first be removed in order to prevent multiple incorporation offirst nucleotides. Enzyme and unincorporated first nucleotides arepreferably removed prior to the ligation step (step (5)).

Removal of the reversible terminator group from the first nucleotide canbe performed by any suitable means. For example, removal can beperformed by the use of a chemical, such as tris(carboxyethyl)phosphine(TCEP).

In step (5) of the method a double-stranded ligation polynucleotide isligated (405) to the cleaved scaffold polynucleotide. The ligationpolynucleotide comprises a support strand and a helper strand. Theligation polynucleotide further comprises a complementary ligation endcomprising in the support strand a universal nucleotide and a singleoverhanging nucleotide which overhang the terminal nucleotide of thehelper strand. The overhanging nucleotide is the partner nucleotide forthe first nucleotide of the predefined sequence. The ligationpolynucleotide further comprises in the helper strand adjacent theoverhang a terminal nucleotide which is configured such that it cannotbe ligated to another polynucleotide strand (position labelled “K” inthe structure depicted at the top left of the upper part of FIG. 4).Typically this terminal nucleotide will lack a phosphate group.Typically, as described above, this terminal nucleotide of the helperstrand will define the 5′ terminus of the helper strand.

In the complementary ligation end of the ligation polynucleotide theuniversal nucleotide in the support strand is positioned (n+3) oppositea nucleotide which is two positions removed from the terminal nucleotideof the helper strand (position labelled “X” in step (5) of FIG. 4) andis paired therewith. The partner nucleotide for the first nucleotide ofthe predefined sequence occupies a position as the terminal nucleotideof the support strand of the ligation polynucleotide (position n). Thenucleotide which is one position removed from the terminal nucleotide ofthe support strand of the ligation polynucleotide is labelled “J” instep (5) of FIG. 4 at position n+1. The nucleotide which is twopositions removed from the terminal nucleotide of the support strand ofthe ligation polynucleotide is labelled “H” in step (5) of FIG. 4 atposition n+2.

The complementary ligation end is configured so that it will compatiblyjoin with the overhanging end of the cleaved scaffold polynucleotideproduct following incorporation step (3) when subjected to suitableligation conditions. Upon ligation of the support strands, the firstnucleotide becomes paired with its partner nucleotide.

Ligation of the support strands may be performed by any suitable means.Ligation will result in the joining of the support strands only, withthe maintenance of a single-stranded break between the first nucleotidein the synthesis strand, i.e. in the primer strand portion, and theterminal nucleotide of the helper strand adjacent the first nucleotide.

Ligation may typically be performed by enzymes having ligase activity.For example, ligation may be performed with T3 DNA ligase or T4 DNAligase or functional variants or equivalents thereof. The use of suchenzymes will result in the maintenance of the single-stranded break inthe synthesis strand, since the terminal nucleotide of the helper strandis provided such that it cannot act as a substrate for ligase, e.g. dueto the absence of a terminal phosphate group.

Ligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide completes a first synthesis cycle whereupon the scaffoldpolynucleotide of step (1) is effectively re-constituted except that thefirst nucleotide of the predefined nucleotide sequence is incorporatedinto the scaffold polynucleotide opposite its partner nucleotide.

In step (5) of this exemplary method (305), in the complementaryligation end of the ligation polynucleotide the universal nucleotide inthe support strand is positioned opposite the nucleotide which is twopositions removed from the terminal nucleotide of the helper strandadjacent the single-strand break site (shown at position “X” in step (5)of FIG. 4). It will be noted that in the context of the ligationpolynucleotide the universal nucleotide is positioned at position n+3and three positions removed from the terminal nucleotide of the supportstrand (position n). Position n in the context of the ligationpolynucleotide has the same meaning as described above in the context ofthe scaffold polynucleotide at steps (1) and (2). Thus in step (5) by“n” it is meant the nucleotide position in the support strand which,following ligation, is opposite the position in the synthesis strandwhich is now occupied by the nucleotide of the predefined sequencefollowing its addition to the terminal end of the primer strand portionof the synthesis strand in that cycle of synthesis. By “n” it is alsomeant the nucleotide position in the synthesis strand following ligationwhich is now occupied by the nucleotide of the predefined sequencefollowing its addition to the terminal end of the primer strand portionof the synthesis strand in that cycle of synthesis.

It will be noted that the universal nucleotide occupies position n+2 inthe scaffold polynucleotide during steps (1) and (2) (401 and 402 ofFIG. 4), whereas the universal nucleotide occupies position n+3 in theligation polynucleotide in the same synthesis cycle (405 of FIG. 4).This is because at step (5) the cleaved double-stranded scaffoldpolynucleotide is now provided with the nucleotide of the predefinedsequence following its addition in that cycle of synthesis and becausethe ligation polynucleotide is provided with the nucleotide to partnerthe nucleotide of the predefined sequence; and furthermore because asdefined herein “n” always refers to the nucleotide position in thesynthesis strand which is occupied by (or will be occupied by) thenucleotide of the predefined sequence incorporated in that cycle orrefers to the nucleotide position opposite thereto in the supportstrand. Thus at the end of any given cycle of synthesis, immediatelyafter ligation step (5), the position occupied by a universal nucleotidewill be n+3 and will have moved from n+2, the position a universalnucleotide will have occupied at step (1). The product of the ligationstep (5) (structure labelled “ligation product” in FIG. 4) will be thescaffold polynucleotide for use in the next cycle of synthesis. When theligation product is considered as the scaffold polynucleotide for use inthe next cycle of synthesis (406) it is to be understood that theposition occupied by the universal nucleotide is now to be once againreferred to as position n+2 (406), rather than n+3 (405). Thus in methodversion 4 the position occupied by the universal nucleotide at thebeginning of any given cycle of synthesis (401, 406 etc.) is alwaysreferred to as position n+2, and the nucleotide which is newlyincorporated in that cycle always occupies a position referred to as n.

Following completion of the first synthesis cycle, second and subsequentcycles are performed using the same method steps.

As in step (2) of the first synthesis cycle of method version 4, in step(6) the scaffold polynucleotide is cleaved (407) at a cleavage site. Thecleavage site is defined by a sequence comprising the universalnucleotide in the support strand. Cleavage results in a double-strandedbreak in the scaffold polynucleotide. Cleavage of the scaffoldpolynucleotide leaves in place a cleaved double-stranded scaffoldpolynucleotide comprising, at the site of cleavage, a cleaved terminalend of the support strand and the terminal end of the primer strandportion of the synthesis strand which comprised the nick site prior tocleavage. Cleavage results in removal of the support strand comprisingthe universal nucleotide and the helper strand hybridised to the supportstrand, if present immediately prior to cleavage.

The cleavage steps may be performed as described above for step (2) ofthe first cycle.

In step (7) the next nucleotide is added to the terminal end of thecleaved primer strand portion of the synthesis strand as in step (3) ofthe first cycle.

In step (8) the reversible terminator group is removed from the nextnucleotide (deprotection step; 409). As described above for first cycle,this can be performed at various stages. Typically, and preferably, itwill be performed as step (8) of the method, before ligation step (9).However, the deprotection step could be performed at any step afterincorporation of the new nucleotide, such as after the ligation step(9).

Deprotection of the reversible terminator group in the next cycle (409)and subsequent cycles may be performed as described above with respectto the first synthesis cycle.

In step (9) of the next cycle a double-stranded ligation polynucleotideis ligated (410) to the cleaved scaffold polynucleotide. The ligationpolynucleotide of step (9) of the next and subsequent synthesis cyclesmay be configured, and the ligation step may be performed, as describedabove for step (5) of the first synthesis cycle.

Synthesis cycles are repeated as described above for as many times asnecessary to synthesise the double-stranded polynucleotide having thepredefined nucleotide sequence.

Variation Methods Based on Synthesis Method Version 4

It will be appreciated that synthesis method version 4 is a variation ofsynthesis method version 2. Both methods require cleavage of the supportstrand of the scaffold polynucleotide between positions n and n−1. Inversion 2 prior to and at the cleavage step of the first and secondcycles (steps 2 and 6) the universal nucleotide occupies position n+1 inthe support strand of the scaffold polynucleotide. In contrast inversion 4 prior to and at the cleavage step of the first and secondcycles (steps 2 and 6) the universal nucleotide occupies position n+2 inthe support strand of the scaffold polynucleotide. Thus synthesis methodversion 4 is the same as synthesis method version 2 except that insynthesis method version 4 the universal nucleotide occupies oneposition further removed from position n in the direction distal toprimer strand portion.

Further variations of synthesis method version 4 are envisaged whereinthe support strand of the scaffold polynucleotide is cleaved betweenpositions n and n−1 and wherein in each further variant method theuniversal nucleotide occupies incrementally one position further removedfrom position n in the direction distal to primer strand portion,starting at position n+3 and increasing incrementally according to theformula n+3+x wherein x is a whole number between 1 to 10 or more.

Thus an “n+3” variation of synthesis method version 4 is providedwherein the method is performed in the same way as synthesis methodversion 4 described above except for the following variations.

Prior to and at the cleavage step of the first cycle (steps 1 and 2) theuniversal nucleotide instead occupies position n+3 in the support strandof the scaffold polynucleotide, wherein n+3 is the third nucleotideposition in the support strand relative to position n in the directionproximal to the helper strand/distal to the primer strand portion; andthe support strand of the scaffold polynucleotide is cleaved betweenpositions n and n−1.

In the ligation step of the first cycle (step 5) the complementaryligation end of the ligation polynucleotide is structured such that theuniversal nucleotide instead occupies position n+4 in the support strandand is paired with the nucleotide which is 3 positions removed from theterminal nucleotide of the helper strand at the complementary ligationend; wherein position n+4 is position 4 in the support strand relativeto position n in the direction proximal to the helper strand/distal tothe primer strand portion.

Prior to and at the cleavage step of the second cycle (steps 5 and 6)and in cleavage steps of all subsequent cycles the universal nucleotideoccupies position n+3 in the support strand of the scaffoldpolynucleotide, and the support strand of the scaffold polynucleotide iscleaved between positions n and n−1.

Finally, in the ligation step of the second cycle (step 9) and inligation steps of all subsequent cycles the complementary ligation endof the ligation polynucleotide is structured such that the universalnucleotide occupies position n+4 in the support strand and is pairedwith the nucleotide which is 2 positions removed from the terminalnucleotide of the helper strand at the complementary ligation end.

In addition to the above-described “n+3” method, “n+3+x” variations ofsynthesis method version 4 are provided wherein the method is performedin the same way as synthesis method version 4 described above except forthe following variations.

Prior to and at the cleavage step of the first cycle (steps 1 and 2) theuniversal nucleotide instead occupies position n+3+x in the supportstrand of the scaffold polynucleotide, wherein n+3 is the thirdnucleotide position in the support strand relative to position n in thedirection proximal to the helper strand/distal to the primer strandportion; and the support strand of the scaffold polynucleotide iscleaved between positions n and n−1.

In the ligation step of the first cycle (step 5) the complementaryligation end of the ligation polynucleotide is structured such that theuniversal nucleotide instead occupies position n+4+x in the supportstrand and is paired with the nucleotide which is 3+x positions removedfrom the terminal nucleotide of the helper strand at the complementaryligation end; wherein position n+4 is position 4 in the support strandrelative to position n in the direction proximal to the helperstrand/distal to the primer strand portion.

Prior to and at the cleavage step of the second cycle (step 6) and incleavage steps of all subsequent cycles the universal nucleotideoccupies position n+3+x in the support strand of the scaffoldpolynucleotide and the support strand of the scaffold polynucleotide iscleaved between positions n and n−1.

Finally, in the ligation step of the second cycle (step 9) and inligation steps of all subsequent cycles the complementary ligation endof the ligation polynucleotide is structured such that the universalnucleotide occupies position n+4+x in the support strand and is pairedwith the nucleotide which is 2+x positions removed from the terminalnucleotide of the helper strand at the complementary ligation end.

In all of these methods, x is a whole number between 1 to 10 or more,and wherein x is the same whole number in steps (1), (2), (5), (6) and(9).

As with synthesis method version 4, with variant methods based onversion 4 it will be noted that the universal nucleotide occupiesposition n+3 (or n+3+x, depending upon the particular variant method) inthe scaffold polynucleotide during steps (1) and (2), whereas theuniversal nucleotide occupies position n+4 (or n+4+x, depending upon theparticular variant method) in the ligation polynucleotide in the samesynthesis cycle. This is because at step (5) the cleaved double-strandedscaffold polynucleotide is now provided with the nucleotide of thepredefined sequence following its addition in that cycle of synthesisand because the ligation polynucleotide is provided with the nucleotideto partner the nucleotide of the predefined sequence; and furthermorebecause as defined herein “n” always refers to the nucleotide positionin the synthesis strand which is occupied by (or will be occupied by)the nucleotide of the predefined sequence incorporated in that cycle orrefers to the nucleotide position opposite thereto in the supportstrand. Thus at the end of any given cycle of synthesis, immediatelyafter ligation step (5), the position occupied by a universal nucleotidewill be n+4 (or n+4+x) and will have moved from n+3 (or correspondinglyfrom n+3+x), the position a universal nucleotide will have occupied atstep (1). The product of the ligation step (5) (corresponding to thestructure labelled “ligation product” in FIG. 4) will be the scaffoldpolynucleotide for use in the next cycle of synthesis. When the ligationproduct is considered as the scaffold polynucleotide for use in the nextcycle of synthesis it is to be understood that the position occupied bythe universal nucleotide is now to be once again referred to as positionn+3 (or n+3+x) (c.f. 406), rather than n+4 (or n+4+x) (c.f. 405). Thusin variants of synthesis method version 4 the position occupied by theuniversal nucleotide at the beginning of any given cycle of synthesis(c.f. 401, 406 etc.) is always referred to as position n+3 (or n+3+xdepending upon the particular variant method, wherein x is a wholenumber between 1 to 10 or more), and the nucleotide which is newlyincorporated in that cycle always occupies a position referred to as n.

Synthesis Method Version 5

A fifth exemplary version of the synthesis method of the invention isperformed in an analogous matter to the first version except withmodified structural arrangements for the positioning of the universalnucleotide relative to the position to be occupied by the new nucleotideto be incorporated in a given synthesis cycle and relative to the nickand cleavage sites.

Thus as with version 1, in version 5 cleavage of the scaffoldpolynucleotide (step 2, 502) results in loss of the helper strand, ifpresent immediately prior to cleavage, and loss of the support strandcomprising the universal nucleotide. Cleavage leaves in place a cleaveddouble-stranded scaffold polynucleotide comprising, at the site ofcleavage, a cleaved terminal end of the support strand and the terminalend of the primer strand portion of the synthesis strand which comprisedthe nick site prior to cleavage. Cleavage results in a cleaveddouble-stranded scaffold polynucleotide with a single nucleotideoverhang at the site of cleavage.

In step (3) of the methods a first nucleotide in the predefinednucleotide sequence is added to the terminal end of the primer strandportion of the synthesis strand by the action of an enzyme havingtransferase activity such as polymerase or a terminal nucleotidyltransferase enzyme (503). The first nucleotide is provided with areversible terminator group which prevents further extension by theenzyme. Thus in step (3) only a single nucleotide is incorporated.

In step (4) of the methods a deprotection step is performed (504) toremove the terminator group from the newly-incorporated nucleotide. Inprinciple the deprotection step may be performed after the ligation step(5). Performance of the deprotection step as step (4) is preferred.

In step (5) of the methods a ligation polynucleotide (see structuredepicted at the top left of the upper part of FIG. 5) is ligated to thecleaved scaffold polynucleotide. Ligation incorporates a partnernucleotide into the scaffold polynucleotide and allows thenewly-incorporated nucleotide to pair with the partner nucleotide (step5 of FIG. 5; 505), thus completing a synthesis cycle.

In the fifth exemplary version of the synthesis method of the inventiona scaffold polynucleotide is provided in step (1) (501) as describedabove. In this method the universal nucleotide in the support strand ofthe scaffold polynucleotide is positioned opposite and paired with anucleotide (labelled “X” in the structure of step (1) of FIG. 5) whichis the penultimate nucleotide of the helper strand adjacent thesingle-strand break site.

In step (2) of the method the scaffold polynucleotide is cleaved (502)at a cleavage site. The cleavage site is defined by a sequencecomprising the universal nucleotide in the support strand. Cleavageresults in a double-stranded break in the scaffold polynucleotide.Cleavage of the scaffold polynucleotide (step 2) results in loss of thehelper strand, if present immediately prior to cleavage, and loss of thesupport strand comprising the universal nucleotide. Cleavage of thescaffold polynucleotide leaves in place a cleaved double-strandedscaffold polynucleotide comprising, at the site of cleavage, a cleavedterminal end of the support strand and the terminal end of the primerstrand portion of the synthesis strand which comprised the nick siteprior to cleavage.

The synthesis strand is already provided with a single-stranded break or“nick” in this exemplary method, thus only cleavage of the supportstrand is necessary to provide a double-stranded break in the scaffoldpolynucleotide.

In this exemplary method version cleavage generates a cleaveddouble-stranded scaffold polynucleotide with a single nucleotideoverhang. Thus the terminal nucleotide of the primer strand portion ofthe synthesis strand overhangs the terminal nucleotide of the supportstrand.

In this method the universal nucleotide occupies position “n+1” in thesupport strand prior to the cleavage step. To obtain such a cleaveddouble-stranded scaffold polynucleotide wherein the terminal nucleotideof the primer strand portion of the synthesis strand overhangs theterminal nucleotide of the support strand when the universal nucleotideoccupies position n+1 in the support strand, the support strand iscleaved at a specific position relative to the universal nucleotide andrelative to the nick site. The support strand of the scaffoldpolynucleotide is cleaved between nucleotide positions n−1 and n−2.

By “n” it is meant the nucleotide position in the support strand whichis opposite the position in the synthesis strand which will be occupiedby the nucleotide of the predefined sequence upon its addition to theterminal end of the cleaved synthesis strand in that cycle of synthesis.By “n” it is also meant the nucleotide position in the synthesis strandwhich will be occupied by the nucleotide of the predefined sequence uponits addition to the terminal end of the primer strand portion of thesynthesis strand in that cycle of synthesis. Thus at the cleavage step,the universal nucleotide is at position n+1 in the support strand, whichis opposite the nucleotide which the penultimate nucleotide of thehelper strand adjacent the single-strand break site (shown at position“X” in step (2) in FIG. 5).

At steps (1) and (2) the position which will be occupied by thenucleotide of the predefined sequence incorporated in that cycle ofsynthesis corresponds with the nucleotide which is the penultimatenucleotide of the helper strand adjacent the single-strand break site(shown at position “X” in step (2) of FIG. 5).

By “n+1” it is meant the next nucleotide position in the support strandrelative to position n in the direction proximal to the helperstrand/distal to the primer strand (nucleotide labelled “Un” at positionn+1, which occupies the position next to the position labelled “H” atposition n in the direction distal to the primer strand, as shownschematically in steps (1) and (2) of FIG. 5). n+1 may also refer to thecorresponding position in the synthesis strand (labelled “X” at positionn+1 in steps (1) and (2) of FIG. 5). Thus at the cleavage step, positionn+1 in the support strand is opposite the position occupied by thepenultimate nucleotide of the helper strand adjacent the nick site.

By “n−1” it is meant the next nucleotide position in the support strandrelative to position n in the direction distal to the helperstrand/proximal to the primer strand (position labelled “J” at positionn−1, as shown schematically in steps (1) and (2) of FIG. 5). n−1 mayalso refer to the corresponding position in the synthesis strand (i.e.the position labelled “K” at position n−1, as shown schematically insteps (1) and (2) of FIG. 5). Thus at the cleavage step, position n−1 inthe support strand is opposite the position occupied by the terminalnucleotide of the primer strand portion of the synthesis strand. Insteps (1) and (2) of FIG. 5 the nucleotides at position “L” and “M” thusoccupy position n−2.

In methods according to version 5, the nucleotide pair which occupypositions H and I; J and K; and L and M can be any nucleotide pair.

Upon cleavage of the support strand between nucleotide positions n−1 andn−2, the universal nucleotide, helper strand (if present) and theportion of the support strand which is or was hybridized to the helperstrand are removed from the remaining cleaved scaffold polynucleotide(see structure depicted at the top right of the upper part of FIG. 5).

A phosphate group should continue to be attached to the terminalnucleotide of the support strand of the cleaved scaffold polynucleotideat the cleavage site (as depicted in the structure shown in the middleof the lower part of FIG. 5). This ensures that the support strand ofthe ligation polynucleotide can be ligated to the support strand of thecleaved scaffold polynucleotide in the ligation step.

Thus in method version 5 the universal nucleotide occupies position n+1in the support strand at steps (1) and (2) and the support strand iscleaved between nucleotide positions n−1 and n−2.

Preferably, the support strand is cleaved by cleavage of thephosphodiester bond between nucleotide positions n−1 and n−2 (the thirdphosphodiester bond of the support strand relative to the position ofthe universal nucleotide, in the direction distal to the helperstrand/proximal to the primer strand).

The support strand may be cleaved by cleavage of one ester bond of thephosphodiester bond between nucleotide positions n−1 and n−2.

Preferably the support strand is cleaved by cleavage of the first esterbond relative to nucleotide position n−1. This will have the effect ofretaining a terminal phosphate group on the support strand of thecleaved scaffold polynucleotide at the cleavage position.

Any suitable mechanism may be employed to effect cleavage of the supportstrand between nucleotide positions n−1 and n−2 when the universalnucleotide occupies position n+1.

Cleavage of the support strand between nucleotide positions n−1 and n−2as described above may be performed by the action of an enzyme.

In this exemplary method version of the invention, as well as with allversions, to allow the next nucleotide to be incorporated in the nextsynthesis cycle, the reversible terminator group must be removed fromthe first nucleotide (deprotection step; 504). This can be performed atvarious stages of the first cycle. Typically, and preferably, it will beperformed as step (4) of the method, before ligation step (5), as shownin step 4 of FIG. 5 (504). However, the deprotection step could beperformed at any step after incorporation of the new nucleotide, such asafter the ligation step (5). Regardless of which stage the deprotectionstep is performed, enzyme and residual unincorporated first nucleotidesshould first be removed in order to prevent multiple incorporation offirst nucleotides. Enzyme and unincorporated first nucleotides arepreferably removed prior to the ligation step (step (5)).

Removal of the reversible terminator group from the first nucleotide canbe performed by any suitable means. For example, removal can beperformed by the use of a chemical, such as tris(carboxyethyl)phosphine(TCEP).

In step (5) of the method a double-stranded ligation polynucleotide isligated (505) to the cleaved scaffold polynucleotide. The ligationpolynucleotide comprises a support strand and a helper strand. Theligation polynucleotide further comprises a complementary ligation endcomprising in the support strand a universal nucleotide and twooverhanging nucleotides which overhang the terminal nucleotide of thehelper strand. The penultimate nucleotide of the support strand of theligation polynucleotide at the complementary ligation end is the partnernucleotide for the first nucleotide of the predefined sequence. Theligation polynucleotide further comprises in the helper strand adjacentthe overhang a terminal nucleotide which is configured such that itcannot be ligated to another polynucleotide strand (position labelled“I” in the structure depicted at the top left of the upper part of FIG.5). Typically this terminal nucleotide will lack a phosphate group.Typically, as described above, this terminal nucleotide of the helperstrand will define the 5′ terminus of the helper strand.

In the complementary ligation end of the ligation polynucleotide theuniversal nucleotide in the support strand is positioned (n+2) oppositea nucleotide which is the penultimate nucleotide of the helper strand(position labelled “X” in step (5) of FIG. 5) and is paired therewith.The terminal nucleotide of the helper strand (position labelled “I” instep (5) of FIG. 5) occupies position n+1 and is shown paired with anucleotide at a position labelled “H” in the support strand and which isalso at position n+1. The partner nucleotide for the first nucleotide ofthe predefined sequence occupies a position as the penultimatenucleotide of the support strand of the ligation polynucleotide atposition n. Finally, the terminal nucleotide of the support strand ofthe ligation polynucleotide is labelled “J” in step (5) of FIG. 5 atposition n−1.

The complementary ligation end is configured so that it will compatiblyjoin with the overhanging end of the cleaved scaffold polynucleotideproduct following incorporation step (3) when subjected to suitableligation conditions. Upon ligation of the support strands, the firstnucleotide becomes paired with its partner nucleotide.

Ligation of the support strands may be performed by any suitable means.Ligation will result in the joining of the support strands only, withthe maintenance of a single-stranded break between the first nucleotidein the synthesis strand, i.e. in the primer strand portion, and theterminal nucleotide of the helper strand adjacent the first nucleotide.

Ligation may typically be performed by enzymes having ligase activity.For example, ligation may be performed with T3 DNA ligase or T4 DNAligase or functional variants or equivalents thereof. The use of suchenzymes will result in the maintenance of the single-stranded break inthe synthesis strand, since the terminal nucleotide of the helper strandis provided such that it cannot act as a substrate for ligase, e.g. dueto the absence of a terminal phosphate group.

Ligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide completes a first synthesis cycle whereupon the scaffoldpolynucleotide of step (1) is effectively re-constituted except that thefirst nucleotide of the predefined nucleotide sequence is incorporatedinto the scaffold polynucleotide opposite its partner nucleotide.

In step (5) of this exemplary method (505), in the complementaryligation end of the ligation polynucleotide the universal nucleotide inthe support strand is positioned opposite the nucleotide which is thepenultimate nucleotide of the helper strand adjacent the single-strandbreak site (shown at position “X” in step (5) of FIG. 5). It will benoted that in the context of the ligation polynucleotide the universalnucleotide is positioned at position n+2 and three positions removedfrom the terminal nucleotide of the support strand (position n−1).Position n in the context of the ligation polynucleotide has the samemeaning as described above in the context of the scaffold polynucleotideat steps (1) and (2). Thus in step (5) by “n” it is meant the nucleotideposition in the support strand which, following ligation, is oppositethe position in the synthesis strand which is now occupied by thenucleotide of the predefined sequence following its addition to theterminal end of the primer strand portion of the synthesis strand inthat cycle of synthesis. By “n” it is also meant the nucleotide positionin the synthesis strand following ligation which is now occupied by thenucleotide of the predefined sequence following its addition to theterminal end of the primer strand portion of the synthesis strand inthat cycle of synthesis.

It will be noted that the universal nucleotide occupies position n+1 inthe scaffold polynucleotide during steps (1) and (2) (501 and 502 ofFIG. 5), whereas the universal nucleotide occupies position n+2 in theligation polynucleotide in the same synthesis cycle (505 of FIG. 5).This is because at step (5) the cleaved double-stranded scaffoldpolynucleotide is now provided with the nucleotide of the predefinedsequence following its addition in that cycle of synthesis and becausethe ligation polynucleotide is provided with the nucleotide to partnerthe nucleotide of the predefined sequence; and furthermore because asdefined herein “n” always refers to the nucleotide position in thesynthesis strand which is occupied by (or will be occupied by) thenucleotide of the predefined sequence incorporated in that cycle orrefers to the nucleotide position opposite thereto in the supportstrand. Thus at the end of any given cycle of synthesis, immediatelyafter ligation step (5), the position occupied by a universal nucleotidewill be n+2 and will have moved from n+1, the position a universalnucleotide will have occupied at step (1). The product of the ligationstep (5) (structure labelled “ligation product” in FIG. 5) will be thescaffold polynucleotide for use in the next cycle of synthesis. When theligation product is considered as the scaffold polynucleotide for use inthe next cycle of synthesis (506) it is to be understood that theposition occupied by the universal nucleotide is now to be once againreferred to as position n+1 (506), rather than n+2 (505). Thus in methodversion 5 the position occupied by the universal nucleotide at thebeginning of any given cycle of synthesis (501, 506 etc.) is alwaysreferred to as position n+1, and the nucleotide which is newlyincorporated in that cycle always occupies a position referred to as n.

Following completion of the first synthesis cycle, second and subsequentcycles are performed using the same method steps.

As in step (2) of the first synthesis cycle of method version 5, in step(6) the scaffold polynucleotide is cleaved (507) at a cleavage site. Thecleavage site is defined by a sequence comprising the universalnucleotide in the support strand. Cleavage results in a double-strandedbreak in the scaffold polynucleotide. Cleavage of the scaffoldpolynucleotide leaves in place a cleaved double-stranded scaffoldpolynucleotide comprising, at the site of cleavage, a cleaved terminalend of the support strand and the terminal end of the primer strandportion of the synthesis strand which comprised the nick site prior tocleavage. Cleavage results in removal of the support strand comprisingthe universal nucleotide and the helper strand hybridised to the supportstrand, if present immediately prior to cleavage.

The cleavage steps may be performed as described above for step (2) ofthe first cycle.

In step (7) the next nucleotide is added to the terminal end of thecleaved primer strand portion of the synthesis strand as in step (3) ofthe first cycle.

In step (8) the reversible terminator group is removed from the nextnucleotide (deprotection step; 509). As described above for first cycle,this can be performed at various stages. Typically, and preferably, itwill be performed as step (8) of the method, before ligation step (9).However, the deprotection step could be performed at any step afterincorporation of the new nucleotide, such as after the ligation step(9).

Deprotection of the reversible terminator group in the next cycle (509)and subsequent cycles may be performed as described above with respectto the first synthesis cycle.

In step (9) of the next cycle a double-stranded ligation polynucleotideis ligated (510) to the cleaved scaffold polynucleotide. The ligationpolynucleotide of step (9) of the next and subsequent synthesis cyclesmay be configured, and the ligation step may be performed, as describedabove for step (5) of the first synthesis cycle.

Synthesis cycles are repeated as described above for as many times asnecessary to synthesise the double-stranded polynucleotide having thepredefined nucleotide sequence.

Variation Methods Based on Synthesis Method Version 5

It will be appreciated that synthesis method version 5 is a variation ofsynthesis method version 3. Both methods require cleavage of the supportstrand of the scaffold polynucleotide between positions n−1 and n−2. Inversion 3 prior to and at the cleavage step of the first and secondcycles (steps 2 and 6) the universal nucleotide occupies position n inthe support strand of the scaffold polynucleotide. In contrast inversion 5 prior to and at the cleavage step of the first and secondcycles (steps 2 and 6) the universal nucleotide occupies position n+1 inthe support strand of the scaffold polynucleotide. Thus synthesis methodversion 5 is the same as synthesis method version 3 except that insynthesis method version 5 the universal nucleotide occupies oneposition further removed from position n in the direction distal toprimer strand portion.

Further variations of synthesis method version 5 are envisaged whereinthe support strand of the scaffold polynucleotide is cleaved betweenpositions n−1 and n−2 and wherein in each further variant method theuniversal nucleotide occupies incrementally one position further removedfrom position n in the direction distal to primer strand portion,starting at position n+2 and increasing incrementally according to theformula n+2+x wherein x is a whole number between 1 to 10 or more.

Thus an “n+2” variation of synthesis method version 5 is providedwherein the method is performed in the same way as synthesis methodversion 5 described above except for the following variations.

In the cleavage step of the first cycle (step 2) the universalnucleotide instead occupies position n+2 in the support strand of thescaffold polynucleotide, wherein n+2 is the second nucleotide positionin the support strand relative to position n in the direction proximalto the helper strand/distal to the primer strand portion; and thesupport strand of the scaffold polynucleotide is cleaved betweenpositions n and n−1.

In the ligation step of the first cycle (step 5) the complementaryligation end of the ligation polynucleotide is structured such that theuniversal nucleotide instead occupies position n+3 in the support strandand is paired with the nucleotide which is 2 positions removed from theterminal nucleotide of the helper strand at the complementary ligationend; wherein position n+3 is position 3 in the support strand relativeto position n in the direction proximal to the helper strand/distal tothe primer strand portion.

In the cleavage step of the second cycle (step 6) and in cleavage stepsof all subsequent cycles the universal nucleotide occupies position n+2in the support strand of the scaffold polynucleotide, and the supportstrand of the scaffold polynucleotide is cleaved between positions n andn−1.

Finally, in the ligation step of the second cycle (step 9) and inligation steps of all subsequent cycles the complementary ligation endof the ligation polynucleotide is structured such that the universalnucleotide occupies position n+3 in the support strand and is pairedwith the nucleotide which is 2 positions removed from the terminalnucleotide of the helper strand at the complementary ligation end.

In addition to the above-described method, “n+2+x” variations ofsynthesis method version 5 are provided wherein the method is performedin the same way as synthesis method version 5 described above except forthe following variations.

In the cleavage step of the first cycle (step 2) the universalnucleotide instead occupies position n+2+x in the support strand of thescaffold polynucleotide, wherein n+2 is the second nucleotide positionin the support strand relative to position n in the direction proximalto the helper strand/distal to the primer strand portion; and thesupport strand of the scaffold polynucleotide is cleaved betweenpositions n and n−1.

In the ligation step of the first cycle (step 5) the complementaryligation end of the ligation polynucleotide is structured such that theuniversal nucleotide instead occupies position n+3+x in the supportstrand and is paired with the nucleotide which is 2+x positions removedfrom the terminal nucleotide of the helper strand at the complementaryligation end; wherein position n+3 is position 3 in the support strandrelative to position n in the direction proximal to the helperstrand/distal to the primer strand portion.

In the cleavage step of the second cycle (step 6) and in cleavage stepsof all subsequent cycles the universal nucleotide occupies positionn+2+x in the support strand of the scaffold polynucleotide and thesupport strand of the scaffold polynucleotide is cleaved betweenpositions n and n−1.

Finally, in the ligation step of the second cycle (step 9) and inligation steps of all subsequent cycles the complementary ligation endof the ligation polynucleotide is structured such that the universalnucleotide occupies position n+3+x in the support strand and is pairedwith the nucleotide which is 2+x positions removed from the terminalnucleotide of the helper strand at the complementary ligation end.

In all of these methods, x is a whole number between 1 to 10 or more,and wherein x is the same whole number in steps (2), (5), (6) and (9).

As with synthesis method version 5, with variant methods based onversion 5 it will be noted that the universal nucleotide occupiesposition n+2 (or n+2+x, depending upon the particular variant method) inthe scaffold polynucleotide during steps (1) and (2), whereas theuniversal nucleotide occupies position n+3 (or n+3+x, depending upon theparticular variant method) in the ligation polynucleotide in the samesynthesis cycle. This is because at step (5) the cleaved double-strandedscaffold polynucleotide is now provided with the nucleotide of thepredefined sequence following its addition in that cycle of synthesisand because the ligation polynucleotide is provided with the nucleotideto partner the nucleotide of the predefined sequence; and furthermorebecause as defined herein “n” always refers to the nucleotide positionin the synthesis strand which is occupied by (or will be occupied by)the nucleotide of the predefined sequence incorporated in that cycle orrefers to the nucleotide position opposite thereto in the supportstrand. Thus at the end of any given cycle of synthesis, immediatelyafter ligation step (5), the position occupied by a universal nucleotidewill be n+3 (or n+3+x) and will have moved from n+2 (or correspondinglyfrom n+2+x), the position a universal nucleotide will have occupied atstep (1). The product of the ligation step (5) (corresponding to thestructure labelled “ligation product” in FIG. 5) will be the scaffoldpolynucleotide for use in the next cycle of synthesis. When the ligationproduct is considered as the scaffold polynucleotide for use in the nextcycle of synthesis it is to be understood that the position occupied bythe universal nucleotide is now to be once again referred to as positionn+2 (or n+2+x) (c.f. 506), rather than n+3 (or n+3+x) (c.f. 505). Thusin variants of synthesis method version 5 the position occupied by theuniversal nucleotide at the beginning of any given cycle of synthesis(c.f. 501, 506 etc.) is always referred to as position n+2 (or n+2+xdepending upon the particular variant method, wherein x is a wholenumber between 1 to 10 or more), and the nucleotide which is newlyincorporated in that cycle always occupies a position referred to as n.

EXAMPLES

The following Examples provide support for the methods for synthesisinga polynucleotide or oligonucleotide according to the invention, as wellas exemplary constructs used in the methods. The Examples do not limitthe invention.

Other than Example 13, the following Examples describe synthesis methodsaccording to reaction schemes which are related to but which are notwithin the scope of the synthesis methods according to the invention.

The Examples demonstrate the ability to perform synthesis reactionswhich involve steps of addition of a nucleotide of a predefined sequenceto the synthesis strand of a scaffold polynucleotide, cleavage of thescaffold polynucleotide at a cleavage site defined by a universalnucleotide and ligation of a ligation polynucleotide which comprises apartner nucleotide for the added nucleotide of the predefined sequenceas well as a new universal nucleotide for use in creating a cleavagesite for use in the next cycle of synthesis. The methods of the presentinvention incorporate these steps in a modified manner. Thus other thanExample 13 the following Examples provide illustrative support for themethods of the invention defined herein. Example 13 provides datarelating to incorporation of 3′-O-modified-dNTPs by Therminator X DNApolymerase using an incorporation step according to methods of theinvention, e.g. synthesis method versions of the invention 1, 2 and 4(FIGS. 1, 2 and 4 respectively).

In the following Examples, and in corresponding FIGS. 12 to 50,references to synthesis method “versions 1, 2 and 3” or “version 1, 2 or3 chemistry” etc. are to be interpreted according to the reactionschematics set out respectively in FIGS. 6, 7 and 8 and not according tothe reaction schematics set out in any of FIGS. 1 to 5 or descriptionsof the same herein. Example 13 and FIG. 51 are to be interpretedaccording to synthesis methods the invention. In particular according tosynthesis methods of the invention 1, 2 and 4 and associated reactionschematics set out in FIGS. 1, 2 and 4, and more particularlyincorporation step 3 of such methods.

Example 1. Synthesis in the Absence of a Helper Strand

This example describes the synthesis of polynucleotides using 4 steps:incorporation of 3′-O-modified dNTPs on partial double-stranded DNA,cleavage, ligation and deprotection, with the first step taking placeopposite a universal nucleotide, in this particular case inosine.

Step 1: Incorporation

The first step describes controlled addition of a 3′-O-protected singlenucleotide to an oligonucleotide by enzymatic incorporation by DNApolymerase (FIG. 12a ).

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis, ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained fromSigma-Aldrich (FIG. 12 h). The stock solutions were prepared at aconcentration of 100 μM.3. Therminator IX DNA polymerase was used that has been engineered byNew England BioLabs with enhanced ability to incorporate 3-O-modifieddNTPs. However, any DNA polymerase that could incorporate modified dNTPscould be used.Two types of reversible terminators were tested:

Methods

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 12.25 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 0.5 μl of 10 μM primer (synthesised strand) (5 pmol, 1 equiv) (SEQ IDNO: 1, FIGS. 12 h) and 0.75 μl of 10 μM template (support strand) (6pmol, 1.5 equiv) (SEQ ID NO: 2, FIG. 12h ) were added to the reactionmixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added. However, any DNA polymerase that could incorporatemodified dNTPs could be used.5. The reaction was incubated for 20 minutes at 65° C.6. The reaction was stopped by addition of TBE-Urea sample buffer(Novex).7. The reaction was separated on polyacrylamide gel (15%) with TBEbuffer and visualized by ChemiDoc MP imaging system (BioRad).

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5minutes using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample was then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

Results

Customised engineered Therminator IX DNA polymerase from New EnglandBioLabs is an efficient DNA polymerase able to incorporate3′-O-modified-dNTPs opposite a universal nucleotide e.g. inosine (FIG.12b-c ).

Efficient incorporation opposite inosine occurred at a temperature of65° C. (FIG. 12d-e ).

Incorporation of 3′-O-modified-dTTPs opposite inosine requires thepresence of Mn²⁺ ions (FIG. 12f-g ). Successful conversion is marked inbold in FIGS. 12 c, e, g and h.

Conclusion

Incorporation of 3-O-modified-dTTPs opposite inosine can be achievedwith particularly high efficiency using customized engineeredTherminator IX DNA polymerase from New England BioLabs, in the presenceof Mn²⁺ ions and at a temperature at 65° C.

Step 2: Cleavage

The second step describes a two-step cleavage of polynucleotides witheither hAAG/Endo VIII or hAAG/chemical base (FIG. 13a ).

Materials and Methods Materials

1. Oligonucleotides utilized in Example 1 were designed in-house andsynthesised by Sigma Aldrich (see table in FIG. 13(e) for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Methods

A cleavage reaction on oligonucleotides was carried out using theprocedure below:

1. A pipette (Gilson) was used to transfer 410 sterile distilled water(ELGA VEOLIA) into a 1.5 ml Eppendorf tube.2. 5 μl of 10× ThermoPol® reaction buffer NEB (20 mM Tris-HCl, 10 mM(NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8) were thenadded into the same Eppendorf tube.3. 1 μl each of oligonucleotides (FIG. 13e ); template (SEQ ID NO: 3) orany fluorescently tagged long oligo strand, primer with T (SEQ ID NO: 4)and control (SEQ ID NO: 5) all at 5 pmols were added into the same tube.4. 1 μl of Human Alkyladenine DNA Glycosylase (hAAG) NEB (10 units/μl)was added into the same tube.5. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for 1 hour.6. Typically after incubation time had elapsed, the reaction wasterminated by enzymatic heat inactivation (i.e. 65° C. for 20 minutes).

Purification under ambient conditions. The sample mixture was purifiedusing the protocol outlined below:

1. 500 μl of buffer PNI QIAGEN (5M guanidinium chloride) was added tothe sample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 50 μl of Buffer EB QIAGEN (10 mM Tris.CL, pH 8.5)was added to the centre of the column membrane and left to stand for 1min at room temperature.7. The tube was then centrifuged at 13000 rpm for 1 minutes. Eluted DNAconcentration was measured and stored at −20° C. for subsequent use.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop one (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop one was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.

Cleavage of the generated abasic site was carried out using theprocedure below:

1. 2 μl (10-100 ng/0) DNA was added into a sterile 1.5 ml Eppendorftube.2. 40 μl (0.2M) NaOH or 1.5 μl Endo VIII NEB (10 units/0) and 5 μl 10×Reaction Buffer NEB (10 mM Tris-HCl, 75 mM NaCl, 1 mM EDTA, pH 8 @ 25°C.) was also added into the same tube and gently mixed by resuspensionand centrifugation at 13000 rpm for 5 sec.3. The resulting mixture was incubated at room temperature for 5 minutesfor the NaOH treated sample while Endo VIII reaction mixture wasincubated at 37° C. for 1 hr.4. After incubation time had elapsed, the reaction mixture was purifiedusing steps 1-7 of purification protocol as outlined above.

Gel Electrophoresis and DNA Visualization:

1. 5 μl of DNA and TBE-Urea sample buffer (Novex) was added into asterile 1.5 ml Eppendorf tube and heated to 95° C. for 2 minutes using aheat thermoblock (Eppendorf).2. The DNA mixtures were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm x 10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. Detection and visualization of DNA in the gel was carried out withChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis wascarried out on the Image lab 2.0 platform.

Results and Conclusion

The cleavage reaction without a helper strand showed a low percentageyield of cleaved to uncleaved DNA ratio of ˜7%:93% (FIG. 13b-d ).

Cleavage results showed that in this specific example, and based on thespecific reagents used, a low yield of cleaved DNA is obtained in theabsence of a helper strand in comparison to the positive control. Inaddition the use of a chemical base for cleavage of the abasic site wasless time-consuming compared to EndoVIII cleavage.

Step 3: Ligation

The third step describes ligation of polynucleotides with DNA ligase inthe absence of a helper strand. A diagrammatic illustration is shown inFIG. 14.

Materials and Methods Materials

1. Oligonucleotides utilized in Example 1 were designed in-house andsynthesised by Sigma Aldrich (see table in FIG. 14c for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Methods

Ligation reaction on oligonucleotides was carried out using theprocedure below:

1. A pipette (Gilson) was used to transfer 160 sterile distilled water(ELGA VEOLIA) into a 1.5 ml Eppendorf tube.2. 10 μl of 2× Quick Ligation Reaction buffer NEB (132 mM Tris-HCl, 20mM MgCl₂, 2 mM dithiothreitol, 2 mM ATP, 15% Polyethylene glycol(PEG6000) and pH 7.6 at 25° C.) was then added into the same Eppendorftube.3. 1 μl each of oligonucleotides (FIG. 14c ); TAMRA or any fluorescentlytagged phosphate strand (SEQ ID NO: 7), primer with T (SEQ ID NO: 8) andinosine strand (SEQ ID NO: 9), all at 5 pmols, was added into the sametube.4. 1 μl of Quick T4 DNA Ligase NEB (400 units/0) was added into the sametube.5. The reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 minutes.6. Typically after incubation time had elapsed, reaction was terminatedwith the addition of TBE-Urea sample Buffer (Novex).7. The reaction mixture was purified using the protocol outlined inpurification steps 1-7 as described above.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop one (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop one was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5), then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.5. Purified DNA was run on a polyacrylamide gel and visualized inaccordance with the procedure in steps 5-8 described above. No change inconditions or reagents was introduced.

Results and Conclusion

In this specific example, and based on the specific reagents used,ligation of oligonucleotides with DNA ligase, in this particular casequick T4 DNA ligase, at room temperature (24° C.) in the absence of ahelper strand results in a reduced amount of ligation product (FIG. 14b).

Example 2. Version 1 Chemistry with a Helper Strand

This example describes the synthesis of polynucleotides using 4 steps:incorporation of 3′-O-modified dNTPs from a nick site, cleavage,ligation and deprotection, with the first step taking place opposite auniversal nucleotide, in this particular case inosine. The method uses ahelper strand which improves the efficiency of the ligation and cleavagesteps.

Step 1: Incorporation

The first step describes controlled addition of 3′-O-protected singlenucleotide to oligonucleotide by enzymatic incorporation using DNApolymerase (FIG. 15a ).

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis. ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained fromSigma-Aldrich. The stock solutions were prepared at a concentration of100 μM. Oligonucleotides are shown in FIG. 15 b.3. Therminator IX DNA polymerase was used that has been engineered byNew England BioLabs with enhanced ability to incorporate 3-O-modifieddNTPs.

Two types of reversible terminators were tested:

3′-O-azidomethyl-dTTP: 3′-O-allyl-dTTP:

Methods

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 10.25 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 0.5 μl of 10 μM primer (5 pmol, 1 equiv) (SEQ ID NO: 10, Table inFIG. 15(b)), 0.75 μl of 10 μM template (6 pmol, 1.5 equiv) (SEQ ID NO:11, Table in FIG. 15(b)), 2 μl of 10 μM of helper strand (SEQ ID NO: 12,Table in FIG. 15(b)) were added to the reaction mixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 20 minutes at 65° C.6. The reaction was stopped by addition of TBE-Urea sample buffer(Novex).7. The reaction was separated on polyacrylamide gel (15%) TBE buffer andvisualized by ChemiDoc MP imaging system (BioRad).

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5minutes using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm x 10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM Boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

The incorporation step can be studied according to the protocoldescribed above.

Step 2: Cleavage

The second step describes a two-step cleavage of polynucleotides witheither hAAG/Endo VIII or hAAG/chemical base (×2) (FIG. 16a ).

Materials and Methods Materials

1. Oligonucleotides utilized in Example 2 were designed in-house andsynthesised by Sigma Aldrich (see FIG. 16f for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Methods

Cleavage reaction on oligonucleotides was carried out using theprocedure below:

1. A pipette (Gilson) was used to transfer 410 sterile distilled water(ELGA VEOLIA) into a 1.5 ml Eppendorf tube.2. 5 μl of 10× ThermoPol® Reaction buffer NEB (20 mM Tris-HCl, 10 mM(NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8) was thenadded into the same Eppendorf tube.3. 1 μl each of oligonucleotides (FIG. 160; template (SEQ ID NO: 13) orany fluorescently tagged long oligo strand, primer with T (SEQ ID NO:14), control (SEQ ID NO: 15) and helper strand (SEQ ID NO: 16), all at 5pmols, were added into the same tube.4. 1 μl of Human Alkyladenine DNA Glycosylase (hAAG) NEB (10 units/0)was added into the same tube.5. In the reaction using alternative base, 1 μl of Human AlkyladenineDNA Glycosylase (hAAG) NEB (100 units/0) was added.6. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for 1 hour.7. Typically after incubation time had elapsed, the reaction wasterminated by enzymatic heat inactivation (i.e. 65° C. for 20 minutes).

Purification under ambient conditions. The sample mixture was purifiedusing the protocol outlined below:

1. 500 0 of buffer PNI QIAGEN (5M guanidinium chloride) was added to thesample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 50 μl of Buffer EB QIAGEN (10 mM Tris.CL, pH 8.5)was added to the centre of the column membrane and left to stand for 1min at room temperature.7. The tube was then centrifuged at 13000 rpm for 1 minute. Eluted DNAconcentration was measured and stored at −20° C. for subsequent use.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop one (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop one was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.

Cleavage of generated abasic site was carried out using the procedurebelow:

1. 2 μl (10-100 ng/μl) DNA was added into a sterile 1.5 ml Eppendorftube.2. 40 μl (0.2M) NaOH or 1.5 μl Endo VIII NEB (10 units/μl) and 5 μl 10×Reaction Buffer NEB (10 mM Tris-HCl, 75 mM NaCl, 1 mM EDTA, pH 8 @ 25°C.) was also added into the same tube and gently mixed by resuspensionand centrifugation at 13000 rpm for 5 sec.3. The resulting mixture was incubated at room temperature for 5 minutesfor the 0.2 M NaOH treated sample while Endo VIII reaction mixture wasincubated at 37° C. for lhr.4. After incubation time had elapsed, the reaction mixture was purifiedusing steps 1-7 of purification protocol as stated above.

Cleavage of generated abasic site using alternative basic chemical wascarried out using the procedure below:

1. 1 μl (10-100 ng/μl) DNA was added into a sterile 1.5 ml Eppendorftube. 2 μl of N,N′ dimethylethylenediamine Sigma (100 mM) which had beenbuffered at room temperature with acetic acid solution sigma (99.8%) topH 7.4 was then added into the same tube.2. 20 μl of sterile distilled water (ELGA VEOLIA) was added into thetube and gently mixed by resuspension and centrifugation at 13000 rpmfor 5 sec.3. The resulting mixture was incubated at 37° C. for 20 minutes.4. After incubation time had elapsed, the reaction mixture was purifiedusing steps 1-7 of the purification protocol stated above.

Gel Electrophoresis and DNA Visualization:

1. 5 μl of DNA and TBE-Urea sample buffer (Novex) was added into asterile 1.5 ml Eppendorf tube and heated to 95° C. for 2 minutes using aheat thermoblock (Eppendorf).2. The DNA mixtures were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. Detection and visualization of DNA in the gel was carried out withChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis wascarried out on the Image lab 2.0 platform.

Results

Cleavage efficiency at a cleavage site comprising a universalnucleotide, in this particular case inosine, by hAAG DNA glycosylase wassignificantly increased from 10% in absence of helper strand to 50% inpresence of helper strand (FIG. 16b ). hAAG and Endonuclease VIII cleaveinosine with lower efficiency (10%) than hAAG and NaOH (50%). Chemicalcleavage using 0.2M NaOH was shown to be preferable for cleavage of APsites than Endonuclease VIII in the described system using nicked DNA(FIG. 16c ). Mild N,N′-dimethylethylenediamine at neutral pH has highefficiency to cleave abasic sites as 0.2M NaOH, and therefore it ispreferable compared with Endonuclease VIII and NaOH (FIGS. 16d-e ).

Conclusion

Three methods were evaluated for cleavage of DNA containing inosine. Onefull enzymatic method—hAAG/Endonuclease VIII, and two methods combiningchemical and enzymatic cleavage—hAAG/NaOH and hAAG/dimethylethylaminewere studied for DNA cleavage in Example 2.

hAAG/NaOH results showed a much higher yield of cleaved DNA (50%) in thepresence of a helper strand in comparison to the absence of a helperstrand (10%). In these specific examples, and based on the specificreagents used, helper strands increase yield of DNA cleavage.

Enzymatic cleavage using Endonuclease VIII as a substitute for NaOH wasless efficient (10%) compared to NaOH (50%) in the presence of a helperstrand.

The inclusion of an alternative mild chemical baseN,N′-dimethylethylenediamine led to high cleavage efficiency of APsites, as efficient as for NaOH, and, together with addition of 10× hAAGenzyme, had a significant increase on cleaved DNA (see FIG. 16e ).

Step 3: Ligation

The third step describes ligation of polynucleotides with DNA ligase inthe presence of a helper strand. A diagrammatic illustration is shown inFIG. 17 a.

Materials and Methods

Materials 1. Oligonucleotides were designed in-house and synthesised bySigma Aldrich (see FIG. 17d for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Methods

Ligation reaction on oligonucleotides was carried out using theprocedure below:

1. A pipette (Gilson) was used to transfer 16 μl sterile distilled water(ELGA VEOLIA) into a 1.5 ml Eppendorf tube.2. 10 μl of 2× Quick Ligation Reaction buffer NEB (132 mM Tris-HCl, 20mM MgCl₂, 2 mM dithiothreitol, 2 mM ATP, 15% Polyethylene glycol(PEG6000) and pH 7.6 at 25° C.) was then added into the same Eppendorftube.3. 1 μl each of oligonucleotides (FIG. 17d ); TAMRA or any fluorescentlytagged phosphate strand (SEQ ID NO: 18), primer with T (SEQ ID NO: 19)and inosine strand (SEQ ID NO: 20) and helper strand (SEQ ID NO: 21),all at of 5 pmols, was added into the same tube.4. 1 μl of Quick T4 DNA Ligase NEB (400 units/μl) was added into thesame tube.5. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 minutes.6. Typically after incubation time had elapsed, reaction was terminatedwith the addition of TBE-Urea sample Buffer (Novex).7. The reaction mixture was purified using the protocol outlined inpurification steps 1-7 as described above.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop one (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop one was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.5. Purified DNA was run on a polyacrylamide gel and visualized inaccordance with the procedure in steps 5-8 above. No change inconditions or reagents was introduced.

Results and Conclusion

In this specific example, and based on the specific reagents used,reduced ligation activity is observed in the absence of a helper strand(FIG. 17b ), whereas ligation proceeds with high efficiency in presenceof a helper strand (FIG. 17c ) and the product is formed in high yield.

Example 3. Version 2 Chemistry with a Helper Strand

This example describes the synthesis of polynucleotides using 4 steps:incorporation of 3′-O-modified dNTPs on partial double-stranded DNA;cleavage, ligation and deprotection with the first step of incorporationtaking place opposite a naturally complementary nucleotide which ispositioned in the support strand adjacent to a universal nucleotide, inthis particular case inosine.

Step 1: Incorporation Materials and Methods Materials

The first step describes controlled addition of 3′-O-protected singlenucleotide to oligonucleotide by enzymatic incorporation by DNApolymerase (FIG. 18a ).

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis. ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained fromSigma-Aldrich (FIG. 18j ). The stock solutions are prepared inconcentration of 100 μM.3. Therminator IX DNA polymerase was used that has been engineered byNew England BioLabs with enhanced ability to incorporate 3-O-modifieddNTPs.3′-O-azidomethyl reversible terminators of all dNTPs were testedindependently for incorporation:

Methods

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 12.25 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 0.5 μl of 10 μM primer (5 pmol, 1 equiv) (SEQ ID NO: 22, FIG. 18j )and 0.75 μl of 10 μM template-A/G/T/C (6 pmol, 1.5 equiv) (SEQ ID NOS:23 to 26, FIG. 18j ) and 1 μl of 10 μM helper strand-T/C/A/G (10 pmol, 2equiv) (SEQ ID NOS: 27 to 30, FIG. 18j ) were added to the reactionmixture.3. 3′-O-modified-dTTP/dCTP/dATP/dGTP (2 μl of 100 μM) and MnCl₂ (1 μl of40 mM) were added.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 20 minutes at 65° C.6. The reaction was stopped by addition of TBE-Urea sample buffer(Novex).7. The reaction was separated on polyacrylamide gel (15%) TBE buffer andvisualized by ChemiDoc MP imaging system (BioRad).

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5minutes using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm x 10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

Results and Conclusions

Regarding the evaluation of the temperature on the incorporation of3-O-azidomethyl-dTTP using Therminator IX DNA polymerase, the resultsindicate that incorporation of 3′-O-azidomethyl-dTTP in the presence ofa helper strand for ligation goes to 90% after 5 minutes. 10% of primerremains unextended after 20 minutes at 37° C. and 47° C.

Therminator IX DNA polymerase at 2 mM Mn²⁺ ions and a temperature of 37°C. provide good conditions for incorporation of 3′-O-modified-dNTPsopposite a complementary base in DNA with high efficiency in thepresence of the helper strand (from the ligation step from the previouscycle).

Step 2: Cleavage

The second step describes a one-step cleavage of polynucleotides withEndonuclease V (FIG. 19a ).

Materials and Methods Materials

1. Oligonucleotides utilized in Example 3 were designed in-house andsynthesised by Sigma Aldrich (see table in FIG. 19d for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Methods

Cleavage reaction on oligonucleotides was carried out using theprocedure below:

1. A pipette (Gilson) was used to transfer 41 μl sterile distilled water(ELGA VEOLIA) into a 1.5 ml Eppendorf tube.2. 5 μl of 10× Reaction Buffer® NEB (50 mM Potassium Acetate, 20 mMTris-acetate, 10 mM Magnesium Acetate, 1 mM DTT, pH 7.9 @ 25° C.) wasthen added into the same Eppendorf tube.3. 1 μl each of oligonucleotides (FIG. 19d ); Template (SEQ ID NO: 31)or any fluorescently tagged long oligo strand, Primer with T (SEQ ID NO:32) and control (SEQ ID NO: 33) and helper strand (SEQ ID NO: 34), allat 5 pmols, were added into the same tube.4. 1 μl of Human Endonuclease V (Endo V) NEB (10 units/0) was added intothe same tube.5. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for lhour.6. Typically after incubation time had elapsed, reaction was terminatedby enzymatic heat inactivation (i.e. 65° C. for 20 minutes).

The sample mixture was purified using the protocol outlined below:

1. 500 μl of buffer PNI QIAGEN (5M guanidinium chloride) was added tothe sample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 50 μl of Buffer EB QIAGEN (10 mM Tris.CL, pH 8.5)was added to the centre of the column membrane and left to stand for 1min at room temperature.7. The tube was then centrifuged at 13000 rpm for 1 minutes. Eluted DNAconcentration was measured and stored at −20° C. for subsequent use.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop one (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop one was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.

Gel Electrophoresis and DNA Visualization:

1. 5 μl of DNA and TBE-Urea sample buffer (Novex) was added into asterile 1.5 ml Eppendorf tube and heated to 95° C. for 2 minutes using aheat thermoblock (Eppendorf).2. The DNA mixtures were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. Detection and visualization of DNA in the gel was carried out withChemidoc MP (BioRad) using Cy3 LEDS. Visualization and analysis wascarried out on the Image lab 2.0 platform.

Results and Conclusions

Cleavage results from Example 3 showed that a significantly high yieldof cleaved DNA could be obtained with Endonuclease V in the presence orabsence of the helper strand (FIG. 19c ).

Step 3: Ligation

The third step describes ligation of polynucleotides with DNA ligase inthe presence of a helper strand. A diagrammatic illustration is shown inFIG. 20 a.

Materials and Methods Materials

1. Oligonucleotides utilized in Example 3 were designed in-house andsynthesised by Sigma Aldrich (see table in FIG. 20b for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Methods

Ligation reaction on oligonucleotides was carried out using theprocedure below

1. A pipette (Gilson) was used to transfer 16 μl sterile distilled water(ELGA VEOLIA) into a 1.5 ml Eppendorf tube.2. 10 μl of 2× Quick Ligation Reaction buffer NEB (132 mM Tris-HCl, 20mM MgCl₂, 2 mM dithiothreitol, 2 mM ATP, 15% Polyethylene glycol(PEG6000) and pH 7.6 at 25° C.) was then added into the same Eppendorftube.3. 1 μl each of oligonucleotides (FIG. 20b ); TAMRA or any fluorescentlytagged phosphate strand (SEQ ID NO: 35), primer with T (SEQ ID NO: 36)and inosine strand (SEQ ID NO: 37) and helper strand (SEQ ID NO: 38) allhaving an amount of 5 pmols was added into the same tube.4. 1 μl of Quick T4 DNA Ligase NEB (400 units/μ1) was added into thesame tube.5. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 minutes.6. Typically after the incubation time had elapsed, the reaction wasterminated with the addition of TBE-Urea sample Buffer (Novex).7. The reaction mixture was purified using the protocol outlined inpurification steps 1-7 as described above.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop one (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop one was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.5. Purified DNA was run on a polyacrylamide gel and visualized inaccordance with the procedure in steps 5-8 described above. No change inconditions or reagents was introduced.

Gel Electrophoresis and DNA Visualization:

1. 5 μl of DNA and TBE-Urea sample buffer (Novex) was added into asterile 1.5 ml Eppendorf tube and heated to 95° C. for 2 minutes using aheat thermoblock (Eppendorf).2. The DNA mixtures were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. Detection and visualization of DNA in the gel was carried out withChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis wascarried out on the Image lab 2.0 platform.

Step 4: Deprotection

Deprotection step (FIG. 21a ) was studied on DNA model bearing3′-O-azidomethyl group that is introduced to DNA by incorporation of3′-O-azidomethyl-dNTPs by Therminator IX DNA polymerase. Deprotectionwas carried out by tris(carboxyethyl)phosphine (TCEP) and monitored byextension reaction when mixture of all natural dNTPs is added to thesolution of the purified deprotected DNA.

Materials and Methods Materials

1. Oligonucleotides utilized in Example 3 were designed in-house andsynthesised by Sigma Aldrich (see FIG. 21i for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).3. Enzymes were purchased from New England BioLabs.

Methods

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂O₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 12.25 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 1 μl of 10 μM primer (10 pmol, 1 equiv) (SEQ ID NO: 39, FIG. 21i )and 1.5 μl of either 10 μM template-A/G/T/C (15 pmol, 1.5 equiv) (SEQ IDNOS: 40 to 43, FIG. 21i ) were added to the reaction mixture.3. 3′-O-modified-dTTP/dCTP/dATP/dGTP (2 μl of 100 μM) and MnCl₂ (1 μl of40 mM) were added.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 5 minutes at 37° C.6. 4 μL of the sample was taken out and mixed with 0.5 ul of 5 mM dNTPmix and allowed to react for 10 minutes for control reaction.7. 40 μL of the 500 mM TCEP in 1M TRIS buffer pH 7.4 was added to thereaction mixture and allowed to react for 10 minutes at 37° C.8. The reaction mixture was purified using QIAGEN Nucleotide removal kiteluting by 20 μL of 1× Thermopol® buffer.9. 1 μL of 5 mM dNTP mix and 1 μL of DeepVent (exo-) DNA polymerase wereadded to the purified reaction mixture and allowed to react 10 minutes.10. The reaction was stopped by addition of TBE-Urea sample buffer(Novex).11. The reaction was separated on polyacrylamide gel (15%) TBE bufferand visualized by ChemiDoc MP imaging system (BioRad).

Results and Conclusion

50 mM TCEP was not sufficient to cleave 3′-O-azidomethyl group with highefficiency on 0.2 μM DNA model (FIG. 21h ). In contrast, 300 mM TCEPsuccessfully cleaved 3′-O-azidomethyl group with 95% efficiency on 0.2μM DNA model (FIG. 21h ).

Example 4. Version 2 Chemistry with Double Hairpin Model

This Example describes the synthesis of polynucleotides using 4 steps ona two-hairpin model: incorporation of 3′-O-modified dNTPs from a nicksite; cleavage, ligation and deprotection with the first step takingplace opposite a naturally complementary nucleotide which is positionedin the support strand adjacent to a universal nucleotide, in thisparticular case inosine.

Step 1: Incorporation

The first step describes controlled addition of 3′-O-protected singlenucleotide to oligonucleotide by enzymatic incorporation by DNApolymerase (FIG. 22a ).

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis. ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained fromSigma-Aldrich (FIG. 22c ). The stock solutions were prepared inconcentration of 100 μM.3. Therminator IX DNA polymerase was used that has been engineered byNew England BioLabs with enhanced ability to incorporate 3-O-modifieddNTPs.3′-O-azidomethyl-dTTP was tested for incorporation:

3′-O-azidomethyl-dTTP:

Method

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂O₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 10.25 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 0.5 μl of 10 μM hairpin oligonucleotide (5 pmol, 1 equiv) (SEQ ID NO:44, FIG. 22c ) was added to the reaction mixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 20 minutes at 65° C.6. The reaction was stopped by addition of TBE-Urea sample buffer(Novex).7. The reaction was separated on polyacrylamide gel (15%) TBE buffer andvisualized by ChemiDoc MP imaging system (BioRad).

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5minutes using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm x 10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

Results

DNA polymerases incorporate 3′-O-modified-dTTPs opposite its naturallycomplementary base in a hairpin construct.

Step 2: Cleavage

The second step describes a one-step cleavage of a hairpin model in thisparticular case with Endonuclease V (FIG. 23a ).

Materials and Methods Materials

1. Oligonucleotides utilized in Example 4 were designed in-house andsynthesised by Sigma Aldrich (see FIG. 23c for sequences).2. The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Methods

Cleavage reaction on hairpin oligonucleotides was carried out using theprocedure below:

1. A pipette (Gilson) was used to transfer 430 sterile distilled water(ELGA VEOLIA) into a 1.5 ml Eppendorf tube.2. 5 μl of 10× Reaction Buffer® NEB (50 mM potassium acetate, 20 mMTris-acetate, 10 mM magnesium acetate, 1 mM DTT, pH 7.9 @ 25° C.) wasthen added into the same Eppendorf tube.3. 1 μl of hairpin oligonucleotide (SEQ ID NO: 45, FIG. 23c ) having anamount of 5 pmols was added into the same tube.4. 1 μl of Human Endonuclease V (Endo V) NEB (30 units/0) was added intothe same tube.5. The reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for lhour.6. Typically after incubation time had elapsed, the reaction wasterminated by enzymatic heat inactivation (i.e. 65° C. for 20 minutes).

The sample mixture was purified using the protocol outlined below:

1. 500 μl of buffer PNI QIAGEN (5M guanidinium chloride) was added tothe sample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 50 μl of Buffer EB QIAGEN (10 mM Tris.CL, pH 8.5)was added to the centre of the column membrane and left to stand for 1min at room temperature.7. The tube was then centrifuged at 13000 rpm for 1 minute. Eluted DNAconcentration was measured and stored at −20° C. for subsequent use.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop One (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop One was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.

Gel Electrophoresis and DNA Visualization:

1. 5 μl of DNA and TBE-Urea sample buffer (Novex) was added into asterile 1.5 ml Eppendorf tube and heated to 95° C. for 2 minutes using aheat ThermoMixer (Eppendorf).2. The DNA mixtures were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. Detection and visualization of DNA in the gel was carried out withChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis wascarried out on the Image lab 2.0 platform.

Results and Conclusion

Cleavage results from Example 4 showed that a significantly high yieldof digested hairpin DNA was obtained with Endonuclease V at 37° C. (FIG.23b ).

Step 3: Ligation

The third step describes ligation of a hairpin model with DNA ligase.Diagrammatic illustration is shown in FIG. 24 a.

Materials and Methods Materials

1. Oligonucleotides utilized in Example 4 were designed in-house andsynthesised by Sigma Aldrich (see FIG. 24c for sequences).

2 The oligonucleotides were diluted to a stock concentration of 100 uMusing sterile distilled water (ELGA VEOLIA).

Method

Ligation reaction on oligonucleotides was carried out using theprocedure below:

1. A pipette (Gilson) was used to transfer 10 (5 pmols) of TAMRA or anyfluorescently tagged phosphate hairpin oligo (SEQ ID NO: 46) into a 1.5ml Eppendorf tube.2. 15 μl (100 pmols) of inosine-containing hairpin construct (SEQ ID NO:47) was then added into the same tube and gently mixed by resuspensionwith a pipette for 3 seconds.3. 40 μl of Blunt/TA DNA Ligase NEB (180 units/0) was added into thesame tube.4. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 minutes.5. Typically after incubation time had elapsed, the reaction wasterminated with the addition of TBE-Urea sample buffer (Novex).6. The reaction mixture was purified using the protocol outlined inpurification steps 1-7 above.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop One (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop One was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.5. Purified DNA was run on a polyacrylamide gel and visualized inaccordance with the procedure in steps 5-8 as described above. No changein conditions or reagents was introduced.

Gel Electrophoresis and DNA Visualization.

1. 5 μl of DNA and TBE-Urea sample buffer (Novex) was added into asterile 1.5 ml Eppendorf tube and heated to 95° C. for 2 minutes using aheat ThermoMixer (Eppendorf).2. The DNA mixtures were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. Detection and visualization of DNA in the gel was carried out withChemiDoc MP (BioRad) using Cy3 LEDS. Visualization and analysis wascarried out on the Image lab 2.0 platform.

Results

Ligation of hairpin oligonucleotides with blunt/TA DNA ligase at roomtemperature (24° C.) in the presence of a helper strand resulted highyield of ligated product. Ligated hairpin oligonucleotide after 1 minuteshowed a high yield of ligated DNA product with a ratio of ˜85%. Theligated hairpin oligonucleotide after 2 minutes showed a high yield ofligated DNA with a ratio of ˜85%. The ligated hairpin oligonucleotideafter 3 minutes showed a high yield of ligated DNA product with a ratioof ˜85%. The ligated hairpin oligonucleotide after 4 minutes showed ahigh yield of ligated DNA product with a ratio of ˜>85% (FIG. 24b ).

Example 5. Version 2 Chemistry—Complete Cycle on Double Hairpin Model

This Example describes the synthesis of polynucleotides using 4 steps ona double hairpin model: incorporation of 3′-O-modified dNTPs from thenick site; cleavage, ligation and deprotection with the first steptaking place opposite a naturally complementary nucleotide which ispositioned in the support strand adjacent to a universal nucleotide, inthis particular case inosine. One end of the hairpin serves as anattachment anchor.

The method starts by controlled addition of a 3′-O-protected singlenucleotide to an oligonucleotide by enzymatic incorporation by DNApolymerase followed by inosine cleavage, ligation and deprotection (FIG.25a ).

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis. ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained fromSigma-Aldrich (FIG. 25c ). The stock solutions are prepared inconcentration of 100 μM.3. Therminator IX DNA polymerase was used that has been engineered byNew England BioLabs with enhanced ability to incorporate 3-O-modifieddNTPs.3′-O-azidomethyl-dTTP was tested for incorporation:

Method

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 12.5 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 2 μl of 10 μM double hairpin model oligonucleotide (20 pmol, 1 equiv)(SEQ ID NO: 48, FIG. 25c ) were added to the reaction mixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 10 minutes at 37° C.6. The aliquot (5 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix was added and allowed to react for 10 minutes. Thereaction was analysed by gel electrophoresis.7. The reaction mixture was purified using the protocol outlined inpurification steps 1-7.8. The DNA sample was eluted by 20 μl of NEB reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into clean Eppendorf tube.9. 1 μl of Human Endonuclease V (Endo V) NEB (30 units/0) was added intothe same tube.10. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for 1 hour.11. After incubation time had elapsed, reaction was terminated byenzymatic heat inactivation (i.e. 65° C. for 20 minutes).12. The aliquot (5 μl) was taken out of the reaction mixture andanalysed on polyacrylamide gel (15%) using TBE buffer and visualized byChemiDoc MP imaging system (BioRad).13. Reaction mixture was purified using the protocol outlined inpurification steps 1-7 above.14. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.15. 10 μl of 100 μM strand for ligation (1 nmol) (SEQ ID NO: 49, FIG.25c ) were added to the reaction mixture.

16. 40 μl of Blunt/TA DNA Ligase NEB (180 units/0) was added into thepurified DNA sample.

17. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 minutes.18. 40 μl of the 500 mM TCEP in 1M TRIS buffer pH 7.4 was added to thereaction mixture and allowed to react for 10 minutes at 37° C.19. The reaction mixture was purified using QIAGEN nucleotide removalkit eluting by 20 μL of 1× Thermopol® buffer.

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5minutes using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 Amps for40 minutes at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop One (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop One was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample onto thepedestal and selecting the measure icon on the touch screen.5. Purified DNA was run on a polyacrylamide gel and visualized inaccordance with the procedure in section 2 steps 5-8. No change inconditions or reagents was introduced.

The sample mixture was purified after each step using the protocoloutlined below:

1. 500 μl of buffer PNI QIAGEN (5M guanidinium chloride) was added tothe sample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 20 μl of appropriate buffer for the reaction wasadded to the centre of the column membrane and left to stand for 1 minat room temperature.7. The tube was then centrifuged at 13000 rpm for 1 minute. Eluted DNAconcentration was measured and stored at −20° C. for subsequent use.

Results

DNA polymerase incorporates 3′-O-modified-dTTPs opposite its naturallycomplementary base in a double hairpin construct (FIG. 25b ).

Example 6. Version 2 Chemistry—Complete Cycle on Single Hairpin ModelUsing Helper Strand

This Example describes the synthesis of polynucleotides using 4 steps onsingle-hairpin model: incorporation of 3′-O-modified dNTPs from nicksite; cleavage, ligation and deprotection with the first step takingplace opposite a naturally complementary base. The DNA synthesis uses ahelper strand in the process.

The method starts by controlled addition of a 3′-O-protected singlenucleotide to an oligonucleotide by enzymatic incorporation by DNApolymerase followed by inosine cleavage, ligation and deprotection (FIG.26a ).

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis. ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained from SigmaAldrich (FIG. 26b ). The stock solutions are prepared in concentrationof 100 μM.3. Therminator IX DNA polymerase was used that has been engineered byNew England BioLabs with enhanced ability to incorporate 3-O-modifieddNTPs.3′-O-azidomethyl-dTTP was tested for incorporation:

3′-O-azidomethyl-dTTP:

Method

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 12.5 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 2 μl of 10 μM Single hairpin model oligonucleotide (20 pmol, 1 equiv)(SEQ ID NO: 50, FIG. 26b ) and Helper strand (30 pmol, 1.5 equiv) (SEQID NO: 51, FIG. 26b ) were added to the reaction mixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 10 minutes at 37° C.6. The aliquot (5 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix was added and allowed to react for 10 minutes. Thereaction was analysed by gel electrophoresis.7. The reaction mixture was purified using the protocol outlined inpurification steps 1-7 above.8. The DNA sample was eluted by 20 μl of NEB reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.9. 1 μl of Human Endonuclease V (Endo V) NEB (30 units/μl) was addedinto the same tube.10. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for 1 hour.11. After incubation time had elapsed, the reaction was terminated byenzymatic heat inactivation (i.e. 65° C. for 20 minutes).12. The aliquot (5 μl) was taken out of the reaction mixture andanalysed on polyacrylamide gel (15%) using TBE buffer and visualized byChemiDoc MP imaging system (BioRad).13. The reaction mixture was purified using the protocol outlined inpurification steps 1-7 above.14. The DNA sample was eluted by 20 μl of NEB reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into clean Eppendorf tube.15. 10 μl of 100 μM strand for ligation (1 nmol) (SEQ ID NO: 52, FIG.26b ) and 10 μl of 100 μM helper strand for ligation (1 nmol) (SEQ IDNO: 53, FIG. 26b ) were added to the reaction mixture.16. 40 μl of Blunt/TA DNA Ligase NEB (180 units/0) was added into thesame tube.17. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 minutes.18. 40 μL of the 500 mM TCEP in 1M TRIS buffer pH 7.4 was added to thereaction mixture and allowed to react for 10 minutes at 37° C.19. The reaction mixture was purified using QIAGEN Nucleotide removalkit eluting by 20 μL of 1×NEB Thermopol® buffer.20. Typically after incubation time had elapsed, reaction was terminatedwith the addition of TBE-Urea sample Buffer (Novex).

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5minutes using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm x 10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 amps for40 minutes at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop One (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop One was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample unto thepedestal and selecting the measure icon on the touch screen.5. Purified DNA was run on a polyacrylamide gel and visualized inaccordance with the procedure noted above in steps 5-8. No change inconditions or reagents was introduced.

The sample mixture was purified after each step using the protocoloutlined below:

1. 500 μl of buffer PNI QIAGEN (5M guanidinium chloride) was added tothe sample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 20 μl of appropriate buffer for the reaction wasadded to the centre of the column membrane and left to stand for 1minute at room temperature.7. The tube was then centrifuged at 13000 rpm for 1 minute. Eluted DNAconcentration was measured and stored at −20° C. for subsequent use.

Example 7. Version 3 Chemistry—Complete Cycle on Double Hairpin Model

This Example describes the synthesis of polynucleotides using 4 steps ona double-hairpin construct model: incorporation of 3′-O-modified dNTPsfrom the nick site; cleavage, ligation and deprotection with the firststep taking place opposite a universal nucleotide, in this particularcase an inosine base.

The method starts by controlled addition of a 3′-O-protected singlenucleotide to an oligonucleotide by enzymatic incorporation by DNApolymerase followed by inosine cleavage, ligation and deprotection (FIG.27a ).

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-housed according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis. ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained fromSigma-Aldrich (FIG. 27b ). The stock solutions are prepared inconcentration of 100 μM.3. Therminator IX DNA polymerase that has been engineered by New EnglandBioLabs has enhanced ability to incorporate 3-O-modified dNTPs.3′-O-azidomethyl-dTTP was tested for incorporation:

Method

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 12.5 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 2 μl of 10 μl double hairpin model oligonucleotide (20 pmol, 1 equiv)(SEQ ID NO: 54, FIG. 27b ) were added to the reaction mixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 10 minutes at 37° C.6. The aliquot (5 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix was added and allowed to react for 10 minutes. Thereaction was analysed by gel electrophoresis.7. The reaction mixture was purified using the protocol outlined inpurification steps 1-7.8. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into clean Eppendorf tube.9. 1 μl of Human Endonuclease V (Endo V) NEB (30 units/μl) was addedinto the same tube.10. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for 1 hour.11. After the incubation time had elapsed, the reaction was terminatedby enzymatic heat inactivation (i.e. 65° C. for 20 minutes).12. The aliquot (5 μl) was taken out of the reaction mixture andanalysed on polyacrylamide gel (15%) using TBE buffer and visualized byChemiDoc MP imaging system (BioRad).13. Reaction mixture was purified using the protocol outlined inpurification steps 1-7 above.14. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.15. 10 μl of 100 μM strand for ligation (1 nmol) (SEQ ID NO: 55, FIG.27b ), were added to the reaction mixture.16. 40 μl of Blunt/TA DNA Ligase NEB (180 units/0) was added into thesame tube.17. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 minutes.18. 40 μL of the 500 mM TCEP in 1M TRIS buffer pH 7.4 was added to thereaction mixture and allowed to react for 10 minutes at 37° C.19. The reaction mixture was purified using QIAGEN Nucleotide removalkit eluting by 20 μL of 1×NEB Thermopol® buffer.20. Typically after incubation time had elapsed, reaction was terminatedwith the addition of TBE-Urea sample Buffer (Novex).

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5minutes using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm×10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place andelectrophoresis performed at the following conditions; 260V, 90 amps for40 minutes at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop One (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop One was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Step 2 was then repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample unto thepedestal and selecting the measure icon on the touch screen.5. Purified DNA was run on a polyacrylamide gel and visualized inaccordance with the procedure in section 2 steps 5-8. No change inconditions or reagents was introduced.

The sample mixture was purified after each step using the protocoloutlined below:

1. 500 μl of buffer PNI QIAGEN (5M guanidinium chloride) was added tothe sample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 20 μl of appropriate buffer for the reaction wasadded to the centre of the column membrane and left to stand for 1 minat room temperature.7. The tube was then centrifuged at 13000 rpm for 1 minutes. Eluted DNAconcentration was measured and stored at −20° C. for subsequent use.

Example 8. Version 2 Chemistry—Complete Two-Cycle Experiment onDouble-Hairpin Model

This example describes a complete two-cycle experiment for the synthesisof polynucleotides using 4 steps on a double-hairpin model:incorporation of 3′-O-modified dNTPs from the nick site; deprotection,cleavage, and ligation with the first step taking place opposite acomplementary base.

The method starts by controlled addition of a 3′-O-protected singlenucleotide to an oligonucleotide by enzymatic incorporation by DNApolymerase followed by deprotection, inosine cleavage and ligation, asdepicted in the reaction schematic for the first cycle shown in FIG. 28a. FIG. 28b shows a reaction schematic for the second cycle.

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis. ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained fromSigma-Aldrich (FIG. 28d ). The stock solutions are prepared inconcentration of 100 μM.3. Therminator IX DNA polymerase that has been engineered by New EnglandBioLabs has enhanced ability to incorporate 3′-O-modified dNTPs.3′-O-azidomethyl-dTTP and 3′-O-azidomethyl-dCTP were used forincorporation:

Method 1^(st) Cycle:

1. 2 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) wasmixed with 12.5 μl of sterile deionized water (ELGA VEOLIA) in 1.5 mlEppendorf tube.2. 2 μl of 10 μM double hairpin model oligonucleotide (20 pmol, 1 equiv)(SEQ ID NO: 56, FIG. 28d ) were added to the reaction mixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded.4. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.5. The reaction was incubated for 10 minutes at 37° C.6. The aliquot (5 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix was added and allowed to react for 10 min. Thereaction was analysed by gel electrophoresis.7. 40 μL of the 500 mM TCEP in 1M TRIS buffer pH=7.4 was added to thereaction mixture and allowed to react for 10 minutes at 37° C.8. The reaction mixture was purified using the protocol outlined inpurification steps 1-7.9. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.10. 1 μL of Human Endonuclease V (Endo V) NEB (30 units/0) was addedinto the same tube.11. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for 1 hour.12. After incubation time had elapsed, the reaction was terminated byenzymatic heat inactivation (i.e. 65° C. for 20 mins).13. The aliquot (5 μl) was taken out of the reaction mixture andanalysed on polyacrylamide gel (15%) using TBE buffer and visualized byChemiDoc MP imaging system (BioRad).14. Reaction mixture was purified by QIAGEN Nucleotide Removal kit usingthe protocol outlined in purification steps 1-7.15. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.16. 10 μl of 100 μM strand for ligation (1 nmol) (SEQ ID NO: 57, FIG.28d ), were added to the reaction mixture.17. 40 μL of Blunt/TA DNA Ligase NEB (180 units/0) was added into thesame tube.18. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 20 mins.19. Reaction mixture was purified by Streptavidin Magnetic Beads kitusing the protocol outlined in purification steps 1-5.20. Unligated oligonucleotide was digested by Lambda Exonuclease.21. Reaction mixture was purified by QIAGEN Nucleotide Removal kit usingthe protocol outlined in purification steps 1-7.22. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.

2^(nd) Cycle:

23. 3′-O-modified-dCTP (2 μl of 100 μM) and MnCl₂ (1 μl of 40 mM) wereadded.24. 1.5 μl of Therminator IX DNA polymerase (15 U, New England BioLabs)was then added.25. The reaction was incubated for 10 minutes at 37° C.26. The aliquot (5 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix was added and reacted for 10 min. The reaction wasanalysed by gel electrophoresis.27. 40 μL of the 500 mM TCEP in 1M TRIS buffer pH=7.4 was added to thereaction mixture and reacted for 10 minutes at 37° C.28. The reaction mixture was purified using the protocol outlined inpurification steps 1-7.29. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.30. 1 μL of Human Endonuclease V (Endo V) NEB (30 units/0) was addedinto the same tube.31. The reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at 37° C.for 1 hour.32. After incubation time had elapsed, the reaction was terminated byenzymatic heat inactivation (i.e. 65° C. for 20 mins).33. The aliquot (5 μl) was taken out of the reaction mixture andanalysed on polyacrylamide gel (15%) using TBE buffer and visualized byChemiDoc MP imaging system (BioRad).34. The reaction mixture was purified using the protocol outlined inpurification steps 1-7.35. The DNA sample was eluted by 20 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into clean Eppendorf tube.36. 10 μl of 100 μM strand for ligation (1 nmol) (SEQ ID NO: 58, FIG.28d ), were added to the reaction mixture.37. 40 μL of Blunt/TA DNA Ligase NEB (180 units/0) was added into thesame tube.38. Reaction mixture was then gently mixed by resuspension with apipette, centrifuged at 13,000 rpm for 5 seconds and incubated at roomtemperature for 10 mins.39. After incubation time had elapsed, the reaction was terminated withthe addition of TBE-Urea sample Buffer (Novex).

Gel Electrophoresis and DNA Visualization:

1. 5 μl of reaction mixture was added to 5 μl of TBE-Urea sample buffer(Novex) in a sterile 1.5 ml Eppendorf tube and heated to 95° C. for 5mins using a heat ThermoMixer (Eppendorf).2. 5 μl of the sample were then loaded into the wells of a 15% TBE-Ureagel 1.0 mm x 10 well (Invitrogen) which contained preheated 1×TBE bufferThermo Scientific (89 mM Tris, 89 mM boric acid and 2 mM EDTA).3. X-cell sure lock module (Novex) was fastened in place and subjectedto electrophoresis by applying the following conditions; 260V, 90 ampsfor 40 mins at room temperature.4. The gel was visualized by ChemiDoc MP (BioRad) using Cy3 LEDS.Visualization and analysis was carried out on the Image lab 2.0platform.

Measurement of purified DNA concentration was determined using theprotocol below:

1. NanoDrop One (Thermo Scientific) was equilibrated by adding 2 μl ofsterile distilled water (ELGA VEOLIA) onto the pedestal.2. After equilibration, the water was gently wiped off using a lint-freelens cleaning tissue (Whatman).3. NanoDrop One was blanked by adding 2 μl of Buffer EB QIAGEN (10 mMTris.CL, pH 8.5). Then step 2 was repeated after blanking.4. DNA concentration was measured by adding 2 μl of the sample unto thepedestal and selecting the measure icon on the touch screen.

The sample mixture was purified by QIAGEN Nucleotide Removal kit usingthe protocol outlined below:

1. 500 μl of buffer PNI QIAGEN (5M guanidinium chloride) was added tothe sample and mixed by gentle resuspension with a pipette.2. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.3. After centrifugation, flow-through was discarded and 750 μl of bufferPE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added into thespin column and centrifuged for 1 min at 6000 rpm.4. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.5. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.6. For DNA elution, 20 μl of appropriate buffer for the reaction wasadded to the centre of the column membrane and left to stand for 1 minat room temperature.7. The tube was then centrifuged at 13000 rpm for 1 min.

After the ligation step, the sample mixture was purified usingStreptavidin Magnetic Beads via the protocol outlined below:

1. 100 μl of Streptavidin Magnetic Beads (New England BioLabs) werewashed 3 times by 200 μl of binding buffer (20 mM TRIS, 500 mM NaCl,pH=7.4).2. Reaction mixture after ligation step is mixed with 10 volumes ofbinding buffer (20 mM TRIS, 500 mM NaCl, pH=7.4) and incubated withStreptavidin Magnetic Beads for 15 minutes at 20° C.3. Streptavidin Magnetic Beads were washed 3 times by 200 μl of bindingbuffer (20 mM TRIS, 500 mM NaCl, pH=7.4).4. Streptavidin Magnetic Beads were washed 3 times by deionized water.5. The oligonucleotides were eluted by 40 μl of deionized water byheating to 95° C. for 3 minutes.

The results shown in FIG. 28c demonstrate the performance two completesynthesis cycles using an exemplary method of the invention.

Example 9. Version 2 Chemistry—Complete Three-Cycle Experiment onSingle-Hairpin Model

This example describes a complete three-cycle experiment for thesynthesis of polynucleotides using 5 steps on a double-hairpin model:incorporation of 3′-O-modified dNTPs from the nick site, deprotection,cleavage, ligation and denaturation step with the first step takingplace opposite a complementary base.

Exemplary schematic overviews of the method are shown in FIGS. 33, 34and 35.

The method starts by the controlled addition of a 3′-O-protected singlenucleotide to an oligonucleotide by enzymatic incorporation by DNApolymerase followed by deprotection, cleavage, ligation, anddenaturation of the helper strand. FIG. 33 shows the 1st full cycleinvolving enzymatic incorporation, deprotection, cleavage, ligation anddenaturation steps. In this example the oligonucleotide is extended by Tnucleotide. FIG. 34 shows the 2nd full cycle following the 1st cycleinvolving enzymatic incorporation, deprotection, cleavage, ligationsteps, and denaturation steps. In this example the oligonucleotide isextended by T nucleotide. FIG. 35 shows the 3rd full cycle following the2nd cycle involving enzymatic incorporation, deprotection, cleavage,ligation, and denaturation steps. In this example the oligonucleotide isextended by T nucleotide.

Materials and Methods Materials

1. 3′-O-modified dNTPs were synthesised in-house according to theprotocol described in PhD thesis Jian Wu: Molecular Engineering of NovelNucleotide Analogues for DNA Sequencing by Synthesis, ColumbiaUniversity, 2008. The protocol for synthesis is also described in thepatent application publication: J. William Efcavitch, Juliesta E.Sylvester, Modified Template-Independent Enzymes for PolydeoxynucleotideSynthesis, Molecular Assemblies US2016/0108382A1.2. Oligonucleotides were designed in house and obtained from IntegratedDNA Technologies, Sigma-Aldrich (FIG. 36). The stock solutions areprepared in concentration of 100 μM.3. Therminator X DNA polymerase was used that has been engineered by NewEngland BioLabs with enhanced ability to incorporate 3-O-modified dNTPs.Any DNA polymerase or other enzyme that could incorporate modified dNTPscould alternatively be used.3′-O-azidomethyl-dTTP was used for incorporation:

Method 1^(st) Cycle:

1. 20 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10mM KCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs) andMnCl₂ solution (10 μl of 40 mM) were mixed with 139 μl of steriledeionized water (ELGA VEOLIA) in 1.5 ml Eppendorf tube.2. 20 μl of 100 μM single hairpin model oligonucleotide (2 nmol, 1equiv) (SEQ ID NO: 59, FIG. 36) was added to the reaction mixture.3. The aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.4. 3′-O-modified-dTTP (10 μl of 2 mM) was added.5. 5 μl of Therminator X DNA polymerase (50 U, New England BioLabs) wasthen added. However, any DNA polymerase or other enzyme that couldincorporate modified dNTPs could be used.6. The reaction was incubated for 30 minutes at 37° C.7. The reaction mixture was purified using QIAGEN Nucleotide Removal kitoutlined in purification steps 66-72.8. The DNA sample was eluted by 200 μl of TE buffer into a cleanEppendorf tube.9. The aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.10. 400 μL of the 500 mM TCEP was added to the reaction mixture andallowed to react for 10 minutes at 37° C.11. The reaction mixture was purified using QIAGEN Nucleotide Removalkit outlined in purification steps 66-72.12. The DNA sample was eluted by 150 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into clean Eppendorf tube.13. The aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.14. 5 μl of Human Endonuclease V (Endo V) NEB (30 units/μ1) was added tothe eluate and incubated at 37° C. for 30 minutes. Any suitablealternative endonuclease could be used.15. After incubation time had elapsed, the reaction was terminated byenzymatic heat inactivation at 65° C. for 20 mins.16. An aliquot (5 μl) was taken out of the reaction mixture and analysedon a polyacrylamide gel.17. The reaction mixture was purified by QIAGEN Nucleotide Removal kitusing the protocol outlined in purification steps 66-72.18. The DNA sample was eluted by 100 μl of T3 DNA ligase buffer (2×concentrate) into a clean Eppendorf tube.19. 20 μl of 100 μM inosine strand for ligation (2 nmol) and 20 μl of100 μM helper strand for ligation (2 nmol) (SEQ ID NO: 60, 51, FIG. 36),and 40 μl of water were added to the reaction mixture.20. 20 μl of T3 DNA Ligase NEB (3000 units/μ1) was added into the sametube (this could include any DNA ligating enzyme) and incubated at roomtemperature for 30 mins.

The reaction mixture was purified using the protocol for StreptavidinMagnetic Beads kit including the denaturation step outlined inpurification steps 73-78.

21. The reaction mixture was purified using the protocol for QIAGENNucleotide Removal kit outlined in purification steps 66-72.22. The DNA sample was eluted by 100 μl of TE buffer into a cleanEppendorf tube.

2^(nd) Cycle:

23. 15 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10mM KCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs),MnCl₂ solution (7.5 μl of 40 mM) and 19 μl of deionized water was added.24. An aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.25. 3′-O-modified-dTTP (7.5 μl of 2 mM) was added.26. 5 μl of Therminator X DNA polymerase (50 U, New England BioLabs) wasthen added. Any DNA polymerase that could incorporate modified dNTPscould be used.27. The reaction was incubated for 30 minutes at 37° C.28. The reaction mixture was purified using QIAGEN Nucleotide Removalkit outlined in purification steps 66-72.29. The DNA sample was eluted by 100 μl of TE buffer into a cleanEppendorf tube.30. An aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.31. 200 μL of the 500 mM TCEP was added to the reaction mixture andallowed to react for 10 minutes at 37° C.32. The reaction mixture was purified using QIAGEN Nucleotide Removalkit outlined in purification steps 66-72.33. The DNA sample was eluted by 100 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.34. The aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.35. 5 μl of Human Endonuclease V (Endo V) NEB (30 units/0) was added tothe eluate, and incubated at 37° C. for 30 minutes. Any suitablealternative endonuclease could be used.36. After incubation time had elapsed, the reaction was terminated byenzymatic heat inactivation at 65° C. for 20 mins.37. The aliquot (5 μl) was taken out of the reaction mixture andanalysed on a polyacrylamide gel.38. The reaction mixture was purified by QIAGEN Nucleotide Removal kitusing the protocol outlined in purification steps 66-72.39. The DNA sample was eluted by 60 μl of T3 DNA ligase buffer (2×concentrate) into a clean Eppendorf tube.40. 20 μl of 100 μM inosine strand for ligation (2 nmol) and 20 μl of100 μM helper strand for ligation (2 nmol) (SEQ ID NO: 60, 51, FIG. 36),and 10 μl of deionized water were added to the reaction mixture.41. 10 μl of T3 DNA Ligase NEB (3000 units/μ1) was added into the sametube and incubated at room temperature for 30 mins. Any suitable DNAligase could be used.42. The reaction mixture was purified using the protocol forStreptavidin Magnetic Beads kit including denaturation step outlined inpurification steps 73-78.43. The reaction mixture was purified using the protocol for QIAGENNucleotide Removal kit outlined in purification steps 66-72.44. The DNA sample was eluted by 46 μl of TE buffer into a cleanEppendorf tube.

3^(rd) Cycle:

45. 6 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10mM KCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England BioLabs),MnCl₂ solution (3 μl of 40 mM) was added.46. An aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.47. 3′-O-modified-dTTP (6 μl of 200 μM) was added.48. 3 μl of Therminator X DNA polymerase (30 U, New England BioLabs) wasthen added. Any DNA polymerase or other suitable enzyme that couldincorporate modified dNTPs could be used.49. The reaction was incubated for 30 minutes at 37° C.50. The reaction mixture was purified using QIAGEN Nucleotide Removalkit outlined in purification steps 66-72.51. The DNA sample was eluted by 50 μl of TE buffer into a cleanEppendorf tube.52. The aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.53. 100 μL of the 500 mM TCEP was added to the reaction mixture andallowed to react for 10 minutes at 37° C.54. The reaction mixture was purified using QIAGEN Nucleotide Removalkit outlined in purification steps 66-72.55. The DNA sample was eluted by 49 μl of NEB Reaction Buffer® (50 mMpotassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mMDTT, pH 7.9 @ 25° C.) into a clean Eppendorf tube.56. An aliquot (4 μl) was taken out of the reaction mixture and 0.5 μlof natural dNTP mix (4 mM) and 0.5 μl of Bst DNA polymerase and 0.5 μlof Sulfolobus DNA polymerase IV were added and allowed to react for 10min. The reaction was analysed by gel electrophoresis.57. 5 μl of Human Endonuclease V (Endo V) NEB (30 units/0) was added tothe eluate and incubated at 37° C. for 30 minutes. Any suitableendonuclease could alternatively be used.58. After incubation time had elapsed, the reaction was terminated byenzymatic heat inactivation at 65° C. for 20 mins.59. The aliquot (5 μl) was taken out of the reaction mixture andanalysed on a polyacrylamide gel.60. The reaction mixture was purified by QIAGEN Nucleotide Removal kitusing the protocol outlined in purification steps 66-72.61. The DNA sample was eluted by 30 μl of T3 DNA ligase buffer (2×concentrate) into a clean Eppendorf tube.62. 10 μl of 100 μM inosine strand for ligation (2 nmol), 10 μl of 100μM helper strand for ligation (2 nmol) (SEQ ID NO: 60, 51, FIG. 36) and5 μl of water were added to the reaction mixture.63. 5 μl of T3 DNA Ligase NEB (3000 units/μ1) was added into the sametube. (This could include any DNA ligating enzyme) and incubated at roomtemperature for 30 mins.64. The reaction mixture was analysed by gel electrophoresis.

Purification of the reaction mixture by QIAGEN Nucleotide Removal kitafter incorporation, deblock and cleavage steps using the protocoloutlined below:

65. 10 volumes of buffer PNI QIAGEN (5M guanidinium chloride) was addedto the sample and mixed by gentle resuspension with a pipette.66. The mixture was transferred into a QIAquick spin column (QIAGEN) andcentrifuged for 1 min at 6000 rpm.67. After centrifugation, flow-through was discarded and 750 μl ofbuffer PE QIAGEN (10 mM Tris-HCl pH 7.5 and 80% ethanol) was added intothe spin column and centrifuged for 1 min at 6000 rpm.68. The flow-through was discarded and the spin column was centrifugedfor an additional 1 min at 13000 rpm to remove residual PE buffer.69. The spin column was then placed in a sterile 1.5 ml Eppendorf tube.70. For DNA elution, 20-200 μl of appropriate buffer for the reactionwas added to the centre of the column membrane and left to stand for 1min at room temperature.71. The tube was then centrifuged at 13000 rpm for 1 min.

Purification of the reaction after the ligation step using StreptavidinMagnetic Beads involving denaturation step was performed via theprotocol outlined below:

72. 100 μl of Streptavidin Magnetic Beads (New England BioLabs) werewashed 3 times by 200 μl of binding buffer (20 mM TRIS, 500 mM NaCl,pH=7.4).73. Reaction mixture after ligation step is mixed with 10 volumes ofbinding buffer (20 mM TRIS, 500 mM NaCl, pH=7.4) and allowed to incubatewith Streptavidin Magnetic Beads for 15 minutes at 20° C.74. Streptavidin Magnetic Beads were washed 3 times by 200 μl of bindingbuffer (20 mM TRIS, 500 mM NaCl, pH=7.4).75. To remove the helper strand, Streptavidin Magnetic Beads were heatedto 80° C. in 200 μl of binding buffer (20 mM TRIS, 500 mM NaCl, pH=7.4),placed to magnet and supernatant was quickly discarded.76. Streptavidin Magnetic Beads were washed 3 times with deionizedwater.77. The oligonucleotides were eluted by 50-100 μl of deionized water byheating to 95° C. for 3 minutes.

Results and Conclusion

FIG. 37 depicts a gel showing reaction products corresponding to a fullthree-cycle experiment comprising: incorporation, deblock, cleavage andligation steps. The results shown demonstrate the performance of threecomplete synthesis cycles using an exemplary method of the invention.

Example 10. Derivatization of a Polyacrylamide Surface and SubsequentImmobilisation of Molecules

This example describes the presentation of bromoacetyl groups on apolyacrylamide surface using N-(5-bromoacetamidylpentyl) acrylamide(BRAPA) and the subsequent surface immobilisation of thiolated moleculesby their covalent coupling to bromoacetyl groups.

Materials and Methods

Glass microscope slides and coverslips were cleaned by ultrasonicationin acetone, ethanol and water sequentially for 10 mins each and driedwith Argon. Clean glass coverslips were silanised withTrichloro(1H,1H,2H,2H-perfluorooctyl)silane in vapor phase in apolystyrene petri dish, sonicated twice in ethanol and dried with Ar(‘fluorinated coverslips’ hereafter). On glass microscope slides, 4%acrylamide/N,N′-Methylenebisacrylamide (19:1) solution was mixed with100 μl of 10% (w/v) ammonium persulphate (APS), 10 μl oftetramethylethylenediamine (TEMED) spiked withN-(5-bromoacetamidylpentyl) acrylamide (BRAPA) at 0, 0.1, 0.2, and 0.3%(w/v) and quickly dispensed into a 4 mm diameter rubber gasket andsubsequently sandwiched with a fluorinated coverslip with thefluorinated side facing towards the acrylamide solution and polymerisedfor 10 mins. After 10 mins, the surfaces were immersed in deionisedwater and left immersed for a total of 4 hrs, during which time thefluorinated coverslips were carefully removed. The polymerisedpolyacrylamide surfaces were dried with Argon.

The polyacrylamide surfaces were subsequently exposed to thiolatedpolyethylene glycol (1 kDa) fluorescein (FITC-PEG-SH), and carboxylatedpolyethylene glycol (1 kDa) fluorescein (FITC-PEG-COOH) as a negativecontrol in sodium phosphate buffer (10 mM, pH 8) for 1 hr andsubsequently washed sequentially with sodium phosphate buffer (10 mM, pH7) and the same buffer containing 0.05% Tween20/0.5M NaCl to eliminatenon-specifically adsorbed thiolated and carboxylated fluorophores. Thesurfaces were subsequently imaged by ChemiDoc (Bio-Rad) in thefluorescein channel.

Results and Conclusion

FIG. 38 shows fluorescence signals and FIG. 39 shown measuredfluorescence from polyacrylamide gel surfaces spiked with differentamount of BRAPA exposed to FITC-PEG-SH and FITC-PEG-COOH. Immobilisationof fluorescein was only successful with polyacrylamide surfaces thatwere spiked with BRAPA and solely with thiolated fluorescein, with closeto zero non-specific adsorption of the carboxylated fluorescein.

Significantly high positive fluorescence signals were obtained frompolyacrylamide surfaces containing BRAPA (BRAPA 0.1, 0.2 and 0.3%) andonly from thiolated molecules (FITC-PEG-SH) compared to thosepolyacrylamide surfaces without BRAPA (BRAPA 0%) and thosepolyacrylamide surfaces containing BRAPA and carboxylated molecules(FITC-PEG-COOH). The results indicate that specific covalent couplinghas occurred between the bromoacetyl moiety from the surface and thethiol moiety from the fluorescein tagged molecules.

The results demonstrate that molecules, such as a molecule comprising asupport strand and a synthesis strand for use in the methods of thepresent invention, can readily be immobilised on a surface substratecompatible with the polynucleotide synthesis reactions described herein.

Example 11. Surface Immobilisation of Hairpin DNA Oligomers andSubsequent Incorporation of Fluorescently Labelled DeoxynucleosideTriphosphates

This example describes:

(1) a method of presenting bromoacetyl groups on a thin polyacrylamidesurface;(2) the subsequent immobilisation of hairpin DNA via covalent couplingof thiophosphate functionalised hairpin DNA with or without a linker;and(3) the incorporation of 2′-deoxynucleotide triphosphate (dNTP) intohairpin DNA.

The method is compatible with virtually any type of material surface(e.g. metals, polymers etc).

(1): Fabrication of a Bromoacetyl Functionalised Thin PolyacrylamideSurface Materials and Methods

Glass microscope slides were first cleaned by ultrasonication in neatDecon 90 (30 mins), water (30 mins), 1M NaOH (15 mins), water (30 mins),0.1M HCl (15 mins), water (30 mins) and finally dried with Argon.

2% (w/v) acrylamide monomer solution was first made by dissolving 1 g ofacrylamide monomer in 50 ml of water. The acrylamide monomer solutionwas vortexed and degassed in argon for 15 mins.N-(5-bromoacetamidylpentyl) acrylamide (BRAPA, 82.5 mg) was dissolved in825 μl of DMF and added to the acrylamide monomer solution and vortexedfurther. Finally, 1 ml of 5% (w/v) potassium persulphate (KPS) and 115μl of neat tetramethylethylenediamine (TEMED) were added to theacrylamide solution, vortexed and the clean glass microscope slides wereexposed to this acrylamide polymerisation mixture for 90 mins. After 90mins, the surfaces were washed with deionised water and dried withargon. These surfaces will be referred to as ‘BRAPA modified surfaces’in this example hereafter. As a negative control, polyacrylamidesurfaces without BRAPA was also made in a similar manner as describedabove by excluding the addition of BRAPA solution into the acrylamidemonomer solution. These surfaces will be referred to as ‘BRAPA controlsurface’ in this example hereafter.

(2): Covalent Coupling of Thiophosphate Functionalised Hairpin DNA ontoPolyacrylamide Surfaces

Materials and Methods

Rubber gaskets with a 4 mm diameter circular opening were placed andsecured onto BRAPA modified and BRAPA control surfaces. The surfaceswere first primed with sodium phosphate buffer (10 mM, pH 7) for 10mins. The buffer was subsequently removed and the surfaces were exposedto 5′-fluorescently labelled (Alexa 647) hairpin DNA oligomers with andwithout a linker modified with six and single thiophosphatesrespectively at a 1 μM concentration and incubated for 1 hr in the dark.BRAPA modified surfaces were also incubated with DNA oligomers with andwithout linker but without thiophosphates as a control (referred to‘oligomer control surfaces’ in this example hereafter). Afterincubation, the surfaces were rinsed in sodium phosphate (100 mM, pH 7)followed by Tris-EDTA buffer (10 mM Tris, 10 mM EDTA, pH 8) and finallywith water. To remove any non-specifically adsorbed DNA oligomers, thesurfaces were subsequently washed with water containing 1M sodiumchloride and 0.05% (v/v) Tween20, washed with water and dried withargon. The surfaces were scanned on ChemiDoc imager in the Alexa 647channel.

FIG. 40a shows the sequences of hairpin DNA without a linker immobilisedon different samples. FIG. 40b shows the sequences of hairpin DNA with alinker immobilised on different samples.

Results

Results are shown in FIGS. 41 and 42. FIG. 41 shows fluorescence signalsoriginating from hairpin DNA oligomers with and without a linkerimmobilised onto bromoacetyl functionalised polyacrylamide surfaces, butnot from BRAPA or oligomer controls.

FIG. 42 shows measured fluorescence intensity following DNAimmobilisation on polyacrylamide surface. The Figure shows the surfacefluorescence signals obtained from various polyacrylamide surfaces andshows that significantly higher signals were obtained from hairpin DNAoligomers immobilised onto BRAPA modified surfaces compared to BRAPA andoligomer control surfaces (as described in (2)), due to successfulcovalent immobilisation of DNA onto bromoacetyl functionalisedpolyacrylamide surfaces.

Conclusion

Fluorescence signals from DNA were only prominently present from BRAPAmodified surfaces that were spiked with BRAPA, indicative of successfulcovalent coupling of DNA onto the surface via the thiophosphatefunctionality. Homogenous and higher signals were obtained from DNA withthe linker compared to DNA without the linker.

(3): Incorporation of Triphosphates into Hairpin DNA Oligomer with aLinker

Materials and Methods

Rubber gaskets with a 9 mm diameter circular opening were placed on theBRAPA modified surfaces immobilised with the DNA oligomer with thelinker and primed with incorporation buffer (50 mM TRIS pH 8, 1 mM EDTA,6 mM MgSO₄, 0.05% tween20 and 2 mM MnCl₂) for 10 mins. The surfaces weresubsequently exposed to incorporation buffer containing DNA polymerase(0.5 U/μl Therminator X DNA polymerase) and triphosphates (20 μM Alexa488 labelled dUTP) and incubated for 1 hr (referred to as ‘polymerasesurface’ in this example hereafter). Additional set of surfaces werealso exposed to incorporation buffer without Therminator X DNApolymerase for 1 hr as a negative control (referred to as ‘negativesurface’ in this example hereafter). After 1 hr, both types of samplewere washed in water, subsequently exposed to water containing 1M sodiumchloride and 0.05% (v/v) Tween20, and washed again with water.Fluorescence signals from the surfaces were measured using ChemiDoc inthe Alexa 647 and Alexa 488 channels to monitor both the presence ofhairpin DNA (Alexa 647) and incorporation of dUTP (Alexa 488).

Results

FIG. 43 shows fluorescence signals detected from Alexa 647 and Alexa 488channels before and after incorporation of Alexa 488-labelled dUTP.Unchanged positive signals from Alexa 647 before and after incorporationindicates that the surface immobilised hairpin DNA is stable during theincorporation reaction, while positive signals from Alexa 488 were onlyobserved from the polymerase surfaces after incorporation reactionshowing the successful incorporation of dUTPs only with the presence ofpolymerase.

FIG. 44 shows measured fluorescence signals in the Alexa 647 (hairpinDNA) and Alexa 488 (dUTP) channels obtained from ‘polymerase surfaces’and ‘negative surfaces’ before and after incorporation of Alexa488-labelled dUTP as described in (3). A significant increase in theAlexa 488 fluorescence signals was obtained after the incorporationreaction from the polymerase surface as a result of the successfulincorporation, while the signals from negative surfaces remained thesame after the incorporation reaction due to the absence of polymerase.Fluorescence signals in the Alexa 647 channel remained virtuallyunchanged after the incorporation reaction, indicating the presence ofhairpin DNA on the surface. The slight reduction in the fluorescencesignal maybe attributed to the effect of photo-bleaching due to thesecond round of light exposure.

Conclusion

The results demonstrate that a molecule comprising a support strand anda synthesis strand for use in the methods of the present invention, canreadily be immobilised on a surface substrate compatible with thepolynucleotide synthesis reactions described herein. The results furtherdemonstrate that such a molecule can accept the incorporation of a newdNTP so as to extend the synthesis strand, whilst at the same time themolecule remains stable and attached to the substrate.

Example 12. Cleavage and Ligation of Hairpin DNA Oligomers Immobilisedto Derivatized Surfaces Via a Linker and Thiophosphate Covalent Linkage

This example describes the covalent coupling to derivatized surfaces ofthiophosphate functionalised hairpin DNA with a linker, followed bycleavage and ligation reactions. The substrate preparation and couplingof hairpin DNA was carried out as described in Example 11.

(1): Cleavage of Immobilised Hairpin DNA Oligomers with a Linker

Materials and Methods

Hairpin DNA was immobilised on surface BRAPA modified surfaces asdescribed in Example 11. Four sets of triplicate surfaces including allthe experimental controls for cleavage and ligation reactions wereprepared. The experimental conditions are described in FIG. 45a . FIG.45b shows the sequences of hairpin DNA immobilised on different samples.

After the DNA immobilisation step, rubber gaskets with a 9 mm diametercircular opening were placed on all surfaces that were immobilised withDNA labelled with Alexa 647 at the 5′ end and primed with 1×NEBuffer 4(50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 1mM DTT, pH 7.9) for 10 mins. Note that for sample D, the immobilisedhairpin DNA does not contain inosine and inosine is replaced by guanine.All the samples were subsequently exposed to either NEBuffer 4containing 1.5 U/μl Endonuclease V (sample A, B and D) or NEBuffer 4without Endonuclease V (sample C) for 1 hr. All the samples weresubsequently washed with 1×T3 DNA Ligase buffer (66 mM Tris-HCl, 10 mMMgCl2, 1 mM ATP, 7.5% PEG6000, 1 mM DTT, pH 7.6), 1×T3 DNA Ligase buffercontaining 1M sodium chloride and 0.05% (v/v) Tween20, washed again with1×T3 DNA Ligase buffer and scanned on ChemiDoc Imager in the Alexa 647channel.

Results

FIG. 46 shows fluorescence signals from hairpin DNA oligomers before andafter cleavage reactions.

FIG. 47 shows measured fluorescence signals before and after cleavagereactions obtained from DNA immobilised surfaces as described above.Successful cleavage reactions were only observed from samples A and B,while fluorescence signal intensities remained almost the same forsamples C and D due to absence of either Endonuclease V (sample C) orinosine in the sequence (sample D).

Significant reductions in the fluorescence signals were observed fromsamples A and B as a result of successful cleavage reactions at theinosine site within the DNA strand with the presence of Endonuclease V.For samples C and D, absence of Endonuclease V and lack of inosine inthe DNA respectively resulted in the fluorescence signals to remainalmost the same level as the initial signals obtained after DNAimmobilisation.

(2): Ligation Reactions Materials and Methods

After the cleavage reaction as described in (1), samples A and B (asdescribed in FIG. 45a ) were exposed to 1×T3 DNA Ligase buffercontaining MnCl₂ (2 mM), inosine strands labelled with Alexa 647 at the5′ end (16 μM) and complimentary ‘helper’ strands (16 μM) (the sequencesare shown in FIG. 48 below) with T3 DNA ligase (250 U/μ1) for sample A,and without T3 DNA Ligase as a negative control for sample B. Sampleswere incubated in the respective solutions for 1 hr. After 1 hr, thesurfaces were washed in water, subsequently exposed to water containing1M sodium chloride and 0.05% (v/v) Tween20, and washed again with water.Fluorescence signals from the surfaces were measured using ChemiDoc inthe Alexa 647 channels. FIG. 48 shows the sequences for theinosine-containing strand and the complimentary ‘helper’ strand forligation reactions.

Results

FIG. 49 shows results relating to the monitoring of ligation reactions.Fluorescence signals detected from Alexa 647 channel before and afterligation reactions. An increase in fluorescence signals in the Alexa 647channels after ligation were only obtained from sample A with T3 DNAligase, while fluorescence signals remained at the same level afterligation reaction for sample B due to the absence of T3 DNA ligase.

FIG. 50 shows that a significant increase in the Alexa 647 fluorescencesignal was obtained after ligation reaction from sample A as a result ofthe successful ligation, where the signal level recovers to the initialsignal level after DNA immobilisation and prior to cleavage reaction asshown in FIG. 47. The fluorescence signals from the sample B remainedthe same after the ligation reaction due to the absence of T3 DNAligase.

Conclusion

The results in this Example demonstrate that a molecule comprising asupport strand and a synthesis strand for use in the methods of thepresent invention, can readily be immobilised on a surface substratecompatible with the polynucleotide synthesis reactions described hereinand can be subjected to cleavage and ligation reactions whilst at thesame time remaining stable and attached to the substrate.

Example 13. Incorporation of 3′-O-azidomethyl-dNTPs to the 3′ terminalEnd of Blunt-Ended DNA

This example describes the incorporation of 3′-O-azidomethyl-dNTPs tothe 3′ end of blunt-ended double-stranded DNA.

The steps below demonstrate the controlled addition of a 3′-O-protectedsingle nucleotide to a blunt-ended double-stranded oligonucleotide byenzymatic incorporation by DNA polymerase. The steps are in accordancewith incorporation step 3 as shown in each of FIGS. 1 and 2 (103, 108,203, 208).

Materials and Methods Materials

-   -   1. In-house synthesized 3′-O-azidomethyl-dNTPs.    -   2. Therminator X DNA polymerase that has been engineered by New        England Biolabs to possess enhanced ability to incorporate        3-O-modified dNTPs.    -   3. Blunt-ended double-stranded DNA oligonucleotide.

Four types of reversible terminators were tested:

Method

1. 5 μl of 10× Thermopol® buffer (20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mMKCl, 2 mM MgSO₄, 0.1% Triton® X-100, pH 8.8, New England Biolabs) wasmixed with 33.5 μl of sterile deionized water (ELGA VEOLIA) in a 1.5 mlEppendorf tube.2. 2 μl of 20 μM primer (40 pmol, 1 equiv) (SEQ ID: NO: 68, FIG. 51a )and 3 μl of 20 μM template (60 pmol, 1.5 equiv) (SEQ ID: NO: 69, FIG.51a ) were added to the reaction mixture.3. 3′-O-modified-dTTP (2 μl of 100 μM) and MnCl₂ (2.5 μl of 40 mM) wereadded.4. 2 μl of Therminator X DNA polymerase (20 U, New England BioLabs) wasthen added.5. The reaction was incubated for 30 minutes at 37° C.6. The reaction was stopped by addition of TBE-Urea sample buffer(Novex).7. The reaction was separated on polyacrylamide gel (15%) TBE buffer andvisualized by ChemiDoc MP imaging system (BioRad).

Results

FIG. 51b depicts a gel showing results of incorporation of3′-O-modified-dNTPs by Therminator X DNA polymerase in the presence ofMn2+ ions at 37° C. The data show that Therminator X DNA polymerase wassuccessfully able to incorporate 3′-O-modified-dNTPs to the 3′ terminalend of the blunt ended DNA oligonucleotide to create a single baseoverhang.

In the above Examples, all oligonucleotides presented in SEQ ID NOS 1-69have a hydroxyl group at the 3′ terminus. All oligonucleotides presentedin SEQ ID NOS 1-69 lack a phosphate group at the 5′ terminus except forSEQ ID NO 7, SEQ ID NO 18 and SEQ ID NO 35.

It is to be understood that different applications of the disclosedmethods and products may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a ligationpolynucleotide” includes two or more such polynucleotides, reference to“a scaffold polynucleotide” includes two or more such scaffoldpolynucleotides, and the like.

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

1. An in vitro method of synthesising a double-stranded polynucleotidehaving a predefined sequence, the method comprising performing cycles ofsynthesis wherein each cycle comprises cleaving a double-strandedpolynucleotide and extending the cleaved double-stranded polynucleotideby incorporating a nucleotide pair, wherein a terminal end of a firststrand of the cleaved double-stranded polynucleotide is extended by theaddition of a nucleotide of the predefined sequence and a terminal endof the second strand of the cleaved double-stranded polynucleotide whichis hybridized to the first strand is extended by the addition of apartner nucleotide thereby forming a nucleotide pair with theincorporated nucleotide of the first strand.
 2. A method according toclaim 1, wherein each cycle comprises extending the first strand byadding the nucleotide of the predefined sequence together with anattached reversible blocking group followed by extending the secondstrand, wherein the reversible blocking group is removed before or afterthe second strand is extended.
 3. A method according to claim 1 or claim2, wherein in each cycle the nucleotides are incorporated into a cleavedscaffold polynucleotide.
 4. A method according to claim 3, wherein eachcycle comprises: (1) providing a scaffold polynucleotide; (2) cleavingthe scaffold polynucleotide at a cleavage site; (3) adding to thecleaved scaffold polynucleotide by the action of a nucleotidetransferase or polymerase enzyme a nucleotide of the predefinedsequence, the nucleotide comprising a reversible terminator group whichprevents further extension by the enzyme; (4) removing the reversibleterminator group from the nucleotide of the predefined sequence; and (5)ligating a ligation polynucleotide to the cleaved scaffoldpolynucleotide, the ligation polynucleotide comprising a partnernucleotide for the nucleotide of the predefined sequence, wherein uponligation the nucleotide of the predefined sequence pairs with thepartner nucleotide.
 5. A method according to claim 4, wherein step (1)comprises providing a scaffold polynucleotide comprising a synthesisstrand and a support strand hybridized thereto, wherein the synthesisstrand comprises a primer strand portion, and the support strandcomprises a universal nucleotide; wherein step (2) comprises cleavingthe scaffold polynucleotide at a cleavage site, the site defined by asequence comprising the universal nucleotide in the support strand,wherein cleavage comprises cleaving the support strand and removing theuniversal nucleotide from the scaffold polynucleotide; and wherein instep (5) the ligation polynucleotide comprises a support strandcomprising the partner nucleotide and a universal nucleotide whichdefines a cleavage site for use in the next cycle, and wherein theligation polynucleotide is ligated to the support strand of the cleavedscaffold polynucleotide thereby forming the nucleotide pair.
 6. A methodaccording to claim 4 or claim 5, the method comprising: (1) providing ascaffold polynucleotide comprising a synthesis strand and a supportstrand hybridized thereto, wherein the synthesis strand comprises aprimer strand portion and a helper strand portion separated by asingle-strand break, and the support strand comprises a universalnucleotide; (2) cleaving the scaffold polynucleotide at a cleavage site,the site defined by a sequence comprising the universal nucleotide inthe support strand, wherein cleavage comprises cleaving the supportstrand and removing the universal nucleotide from the scaffoldpolynucleotide to provide a cleaved double-stranded scaffoldpolynucleotide comprising a support strand and a synthesis strandcomprising the primer strand portion; (3) extending the terminal end ofthe primer strand portion of the synthesis strand of the cleaveddouble-stranded scaffold polynucleotide with a first nucleotide of thepredefined sequence by the action of a nucleotide transferase orpolymerase enzyme, the first nucleotide comprising a reversibleterminator group which prevents further extension by the enzyme; (4)removing the terminator group from the first nucleotide; (5) ligating adouble-stranded ligation polynucleotide to the cleaved scaffoldpolynucleotide, the ligation polynucleotide comprising a support strandand a helper strand hybridised thereto and further comprising acomplementary ligation end, the ligation end comprising: (i) in thesupport strand a universal nucleotide and a partner nucleotide for thefirst nucleotide, wherein the partner nucleotide for the firstnucleotide overhangs the helper strand; and (ii) in the helper strand aterminal nucleotide lacking a phosphate group;  wherein upon ligation ofthe support strands the first nucleotide pairs with the partnernucleotide; (6) cleaving the scaffold polynucleotide at a cleavage site,the site defined by a sequence comprising the universal nucleotide inthe support strand, wherein cleavage comprises cleaving the supportstrand and removing the universal nucleotide from the scaffoldpolynucleotide to provide a cleaved double-stranded scaffoldpolynucleotide comprising a support strand and a synthesis strandcomprising a primer strand portion; (7) extending the terminal end ofthe primer strand portion of the synthesis strand of the cleaveddouble-stranded scaffold polynucleotide with the next nucleotide of thepredefined nucleotide sequence by the action of a nucleotide transferaseor polymerase enzyme, the next nucleotide comprising a reversibleterminator group which prevents further extension by the enzyme; (8)removing the terminator group from the next nucleotide; and (9) ligatinga double-stranded ligation polynucleotide to the cleaved scaffoldpolynucleotide, the ligation polynucleotide comprising a support strandand a helper strand hybridised thereto and further comprising acomplementary ligation end, the ligation end comprising: (i) in thesupport strand a universal nucleotide and a partner nucleotide for thenext nucleotide, wherein the partner nucleotide for the next nucleotideoverhangs the helper strand; and (ii) in the helper strand a terminalnucleotide lacking a phosphate group;  wherein upon ligation of thesupport strands the next nucleotide pairs with the partner nucleotide;(10) repeating steps 6 to 9 multiple times to provide thedouble-stranded polynucleotide having a predefined nucleotide sequence.7. A method according to claim 6, wherein: a) prior to and at thecleavage step of the first cycle (step 2) the universal nucleotideoccupies position n in the support strand of the scaffoldpolynucleotide, wherein position n is the nucleotide position in thesupport strand which is opposite the position in the synthesis strandwhich will be occupied by the first nucleotide of the predefinedsequence upon its addition to the terminal end of the primer strandportion in that cycle, wherein the nucleotide at position n in thesupport strand is opposite the terminal nucleotide of the helper strandand is paired therewith; b) in the cleavage step of the first cycle(step 2) the support strand of the scaffold polynucleotide is cleavedbetween positions n and n−1, wherein position n−1 is the next nucleotideposition in the support strand relative to position n in the directiondistal to the helper strand/proximal to the primer strand portion; c) inthe ligation step of the first cycle (step 5) the complementary ligationend of the ligation polynucleotide is structured such that the partnernucleotide for the first nucleotide of the predefined sequence is theterminal nucleotide of the support strand and occupies position n,wherein the universal nucleotide occupies position n+1 in the supportstrand and is paired with the terminal nucleotide of the helper strand,wherein position n is the nucleotide position which will be opposite thefirst nucleotide of the predefined sequence upon ligation of theligation polynucleotide to the cleaved scaffold polynucleotide in step5; d) in the cleavage step of the second cycle (step 6) and in cleavagesteps of all subsequent cycles: i. the universal nucleotide occupiesposition n in the support strand of the scaffold polynucleotide, whereinposition n is the nucleotide position in the support strand which isopposite the position in the synthesis strand which will be occupied bythe next nucleotide of the predefined sequence upon its addition to theterminal end of the primer strand portion in that cycle; and ii. thesupport strand of the scaffold polynucleotide is cleaved betweenpositions n and n−1, wherein n−1 is the next nucleotide position in thesupport strand relative to position n in the direction distal to thehelper strand/proximal to the primer strand portion; and e) in theligation step of the second cycle (step 9) and in ligation steps of allsubsequent cycles the complementary ligation end of the ligationpolynucleotide is structured such that the partner nucleotide for thenext nucleotide of the predefined sequence in that cycle is the terminalnucleotide of the support strand and occupies position n, and theuniversal nucleotide occupies position n+1 in the support strand and ispaired with the terminal nucleotide of the helper strand; whereinposition n is the nucleotide position which upon ligation of theligation polynucleotide to the cleaved scaffold polynucleotide will beopposite the next nucleotide of the predefined sequence incorporated inthat cycle (step 7).
 8. A method according to claim 6, wherein: a) priorto and at the cleavage step of the first cycle (step 2) the universalnucleotide occupies position n+1 in the support strand of the scaffoldpolynucleotide, wherein position n is the nucleotide position in thesupport strand which is opposite the position in the synthesis strandwhich will be occupied by the first nucleotide of the predefinedsequence upon its addition to the terminal end of the primer strandportion in that cycle, wherein the nucleotide at position n in thesupport strand is opposite the terminal nucleotide of the helper strandand is paired therewith, and wherein n+1 is the next nucleotide positionin the support strand relative to position n in the direction proximalto the helper strand/distal to the primer strand portion; b) in thecleavage step of the first cycle (step 2) the support strand of thescaffold polynucleotide is cleaved between positions n and n−1, whereinposition n−1 is the next nucleotide position in the support strandrelative to position n in the direction distal to the helperstrand/proximal to the primer strand portion; c) in the ligation step ofthe first cycle (step 5) the complementary ligation end of the ligationpolynucleotide is structured such that the partner nucleotide for thefirst nucleotide of the predefined sequence is the terminal nucleotideof the support strand and occupies position n, wherein the universalnucleotide occupies position n+2 in the support strand and is pairedwith the penultimate nucleotide of the helper strand, wherein position nis the nucleotide position which will be opposite the first nucleotideof the predefined sequence upon ligation of the ligation polynucleotideto the cleaved scaffold polynucleotide in step 5 and position n+2 is thesecond position in the support strand relative to position n in thedirection proximal to the helper strand/distal to the primer strandportion; d) in the cleavage step of the second cycle (step 6) and incleavage steps of all subsequent cycles: i. the universal nucleotideoccupies position n+1 in the support strand of the scaffoldpolynucleotide, wherein position n is the nucleotide position in thesupport strand which is opposite the position in the synthesis strandwhich will be occupied by the next nucleotide of the predefined sequenceupon its addition to the terminal end of the primer strand portion inthat cycle, and wherein n+1 is the next nucleotide position in thesupport strand relative to position n in the direction proximal to thehelper strand/distal to the primer strand portion; and ii. the supportstrand of the scaffold polynucleotide is cleaved between positions n andn−1, wherein n−1 is the next nucleotide position in the support strandrelative to position n in the direction distal to the helperstrand/proximal to the primer strand portion; and e) in the ligationstep of the second cycle (step 9) and in ligation steps of allsubsequent cycles the complementary ligation end of the ligationpolynucleotide is structured such that the partner nucleotide for thenext nucleotide of the predefined sequence in that cycle is the terminalnucleotide of the support strand and occupies position n, and theuniversal nucleotide occupies position n+2 in the support strand and ispaired with the penultimate nucleotide of the helper strand; whereinposition n is the nucleotide position which upon ligation of theligation polynucleotide to the cleaved scaffold polynucleotide will beopposite the next nucleotide of the predefined sequence incorporated inthat cycle (step 7) and position n+2 is the second position in thesupport strand relative to position n in the direction proximal to thehelper strand/distal to the primer strand portion.
 9. A method accordingto claim 6, wherein: a) prior to and at the cleavage step of the firstcycle (step 2) the universal nucleotide occupies position n in thesupport strand of the scaffold polynucleotide, wherein position n is thenucleotide position in the support strand which is opposite the positionin the synthesis strand which will be occupied by the first nucleotideof the predefined sequence upon its addition to the terminal end of theprimer strand portion in that cycle, wherein the nucleotide at positionn in the support strand is opposite the terminal nucleotide of thehelper strand and is paired therewith; b) in the cleavage step of thefirst cycle (step 2) the support strand of the scaffold polynucleotideis cleaved between positions n−1 and n−2, wherein positions n−1 and n−2are respectively the next and subsequent nucleotide positions in thesupport strand relative to position n in the direction distal to thehelper strand/proximal to the primer strand portion; c) in the ligationstep of the first cycle (step 5) the complementary ligation end of theligation polynucleotide is structured such that the partner nucleotidefor the first nucleotide of the predefined sequence is the penultimatenucleotide of the support strand and occupies position n, wherein theuniversal nucleotide occupies position n+1 in the support strand and ispaired with the terminal nucleotide of the helper strand, whereinposition n is the nucleotide position which will be opposite the firstnucleotide of the predefined sequence upon ligation of the ligationpolynucleotide to the cleaved scaffold polynucleotide in step 5, andwherein position n+1 is the next nucleotide position in the supportstrand relative to position n in the direction proximal to the helperstrand/distal to the primer strand portion; d) in the cleavage step ofthe second cycle (step 6) and in cleavage steps of all subsequentcycles: i. the universal nucleotide occupies position n in the supportstrand of the scaffold polynucleotide, wherein position n is thenucleotide position in the support strand which is opposite the positionin the synthesis strand which will be occupied by the next nucleotide ofthe predefined sequence upon its addition to the terminal end of theprimer strand portion in that cycle; and ii. the support strand of thescaffold polynucleotide is cleaved between positions n−1 and n−2,wherein positions n−1 and n−2 are respectively the next and subsequentnucleotide positions in the support strand relative to position n in thedirection distal to the helper strand/proximal to the primer strandportion; and e) in the ligation step of the second cycle (step 9) and inligation steps of all subsequent cycles the complementary ligation endof the ligation polynucleotide is structured such that the partnernucleotide for the next nucleotide of the predefined sequence in thatcycle is the penultimate nucleotide of the support strand and occupiesposition n, and the universal nucleotide occupies position n+1 in thesupport strand and is paired with the terminal nucleotide of the helperstrand; wherein position n is the nucleotide position which uponligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide will be opposite the next nucleotide of the predefinedsequence incorporated in that cycle (step 7), and wherein position n+1is the next nucleotide position in the support strand relative toposition n in the direction proximal to the helper strand/distal to theprimer strand portion.
 10. A method according to claim 6, wherein: a)prior to and at the cleavage step of the first cycle (step 2) theuniversal nucleotide occupies position n+2 in the support strand of thescaffold polynucleotide, wherein position n is the nucleotide positionin the support strand which is opposite the position in the synthesisstrand which will be occupied by the first nucleotide of the predefinedsequence upon its addition to the terminal end of the primer strandportion in that cycle, wherein the nucleotide at position n in thesupport strand is opposite the terminal nucleotide of the helper strandand is paired therewith, and wherein n+2 is the second nucleotideposition in the support strand relative to position n in the directionproximal to the helper strand/distal to the primer strand portion; b) inthe cleavage step of the first cycle (step 2) the support strand of thescaffold polynucleotide is cleaved between positions n and n−1, whereinposition n−1 is the next nucleotide position in the support strandrelative to position n in the direction distal to the helperstrand/proximal to the primer strand portion; c) in the ligation step ofthe first cycle (step 5) the complementary ligation end of the ligationpolynucleotide is structured such that the partner nucleotide for thefirst nucleotide of the predefined sequence is the terminal nucleotideof the support strand and occupies position n, wherein the universalnucleotide occupies position n+3 in the support strand and is pairedwith the nucleotide which is two positions removed from the terminalnucleotide of the helper strand in the direction distal to the primerstrand portion, wherein position n is the nucleotide position which willbe opposite the first nucleotide of the predefined sequence uponligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide in step 5 and position n+3 is the third position in thesupport strand relative to position n in the direction proximal to thehelper strand/distal to the primer strand portion; d) in the cleavagestep of the second cycle (step 6) and in cleavage steps of allsubsequent cycles: i. the universal nucleotide occupies position n+2 inthe support strand of the scaffold polynucleotide, wherein position n isthe nucleotide position in the support strand which is opposite theposition in the synthesis strand which will be occupied by the nextnucleotide of the predefined sequence upon its addition to the terminalend of the primer strand portion in that cycle, and wherein n+2 is thesecond nucleotide position in the support strand relative to position nin the direction proximal to the helper strand/distal to the primerstrand portion; and ii. the support strand of the scaffoldpolynucleotide is cleaved between positions n and n−1, wherein positionn−1 is the next nucleotide position in the support strand relative toposition n in the direction distal to the helper strand/proximal to theprimer strand portion; and e) in the ligation step of the second cycle(step 9) and in ligation steps of all subsequent cycles thecomplementary ligation end of the ligation polynucleotide is structuredsuch that the partner nucleotide for the next nucleotide of thepredefined sequence in that cycle is the terminal nucleotide of thesupport strand and occupies position n, and the universal nucleotideoccupies position n+3 in the support strand and is paired with thenucleotide which is two positions removed from the terminal nucleotideof the helper strand in the direction distal to the primer strandportion; wherein position n is the nucleotide position which uponligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide will be opposite the next nucleotide of the predefinedsequence incorporated in that cycle (step 7) and position n+3 is thethird position in the support strand relative to position n in thedirection proximal to the helper strand/distal to the primer strandportion.
 11. A method according to claim 10, wherein: (i) in thecleavage step of the first cycle (step 2) the universal nucleotideinstead occupies position n+3 in the support strand of the scaffoldpolynucleotide, wherein n+3 is the third nucleotide position in thesupport strand relative to position n in the direction proximal to thehelper strand/distal to the primer strand portion; and the supportstrand of the scaffold polynucleotide is cleaved between positions n andn−1; (ii) in the ligation step of the first cycle (step 5) thecomplementary ligation end of the ligation polynucleotide is structuredsuch that the universal nucleotide instead occupies position n+4 in thesupport strand and is paired with the nucleotide which is 3 positionsremoved from the terminal nucleotide of the helper strand at thecomplementary ligation end; wherein position n+4 is position 4 in thesupport strand relative to position n in the direction proximal to thehelper strand/distal to the primer strand portion; (iii) in the cleavagestep of the second cycle (step 6) and in cleavage steps of allsubsequent cycles the universal nucleotide occupies position n+3 in thesupport strand of the scaffold polynucleotide, and the support strand ofthe scaffold polynucleotide is cleaved between positions n and n−1; and(iv) in the ligation step of the second cycle (step 9) and in ligationsteps of all subsequent cycles the complementary ligation end of theligation polynucleotide is structured such that the universal nucleotideoccupies position n+4 in the support strand and is paired with thenucleotide which is 2 positions removed from the terminal nucleotide ofthe helper strand at the complementary ligation end.
 12. A methodaccording to claim 10, wherein: (i) in the cleavage step of the firstcycle (step 2) the universal nucleotide instead occupies position n+3+xin the support strand of the scaffold polynucleotide, wherein n+3 is thethird nucleotide position in the support strand relative to position nin the direction proximal to the helper strand/distal to the primerstrand portion; and the support strand of the scaffold polynucleotide iscleaved between positions n and n−1; (ii) in the ligation step of thefirst cycle (step 5) the complementary ligation end of the ligationpolynucleotide is structured such that the universal nucleotide insteadoccupies position n+4+x in the support strand and is paired with thenucleotide which is 3+x positions removed from the terminal nucleotideof the helper strand at the complementary ligation end; wherein positionn+4 is position 4 in the support strand relative to position n in thedirection proximal to the helper strand/distal to the primer strandportion; (iii) in the cleavage step of the second cycle (step 6) and incleavage steps of all subsequent cycles the universal nucleotideoccupies position n+3+x in the support strand of the scaffoldpolynucleotide and the support strand of the scaffold polynucleotide iscleaved between positions n and n−1; (iv) in the ligation step of thesecond cycle (step 9) and in ligation steps of all subsequent cycles thecomplementary ligation end of the ligation polynucleotide is structuredsuch that the universal nucleotide occupies position n+4+x in thesupport strand and is paired with the nucleotide which is 2+x positionsremoved from the terminal nucleotide of the helper strand at thecomplementary ligation end; and (v) wherein x is a whole number between1 to 10 or more, and wherein x is the same whole number in steps (2),(5), (6) and (9).
 13. A method according to claim 6, wherein: a) priorto and at the cleavage step of the first cycle (step 2) the universalnucleotide occupies position n+1 in the support strand of the scaffoldpolynucleotide, wherein position n is the nucleotide position in thesupport strand which is opposite the position in the synthesis strandwhich will be occupied by the first nucleotide of the predefinedsequence upon its addition to the terminal end of the primer strandportion in that cycle, wherein the nucleotide at position n in thesupport strand is opposite the terminal nucleotide of the helper strandand is paired therewith, and wherein n+1 is the next nucleotide positionin the support strand relative to position n in the direction proximalto the helper strand/distal to the primer strand portion; b) in thecleavage step of the first cycle (step 2) the support strand of thescaffold polynucleotide is cleaved between positions n−1 and n−2,wherein positions n−1 and n−2 are respectively the next and subsequentnucleotide positions in the support strand relative to position n in thedirection distal to the helper strand/proximal to the primer strandportion; c) in the ligation step of the first cycle (step 5) thecomplementary ligation end of the ligation polynucleotide is structuredsuch that the partner nucleotide for the first nucleotide of thepredefined sequence is the penultimate nucleotide of the support strandand occupies position n, wherein the universal nucleotide occupiesposition n+2 in the support strand and is paired with the penultimatenucleotide of the helper strand, wherein position n is the nucleotideposition which will be opposite the first nucleotide of the predefinedsequence upon ligation of the ligation polynucleotide to the cleavedscaffold polynucleotide, and wherein position n+2 is the secondnucleotide position in the support strand relative to position n in thedirection proximal to the helper strand/distal to the primer strandportion; d) in the cleavage step of the second cycle (step 6) and incleavage steps of all subsequent cycles: i. the universal nucleotideoccupies position n+1 in the support strand of the scaffoldpolynucleotide, wherein position n is the nucleotide position in thesupport strand which is opposite the position in the synthesis strandwhich will be occupied by the next nucleotide of the predefined sequenceupon its addition to the terminal end of the primer strand portion inthat cycle, and wherein n+1 is the next nucleotide position in thesupport strand relative to position n in the direction proximal to thehelper strand/distal to the primer strand portion; and ii. the supportstrand of the scaffold polynucleotide is cleaved between positions n−1and n−2, wherein positions n−1 and n−2 are respectively the next andsubsequent nucleotide positions in the support strand relative toposition n in the direction distal to the helper strand/proximal to theprimer strand portion; e) in the ligation step of the second cycle (step9) and in ligation steps of all subsequent cycles the complementaryligation end of the ligation polynucleotide is structured such that thepartner nucleotide for the next nucleotide of the predefined sequence inthat cycle is the penultimate nucleotide of the support strand andoccupies position n, and the universal nucleotide occupies position n+2in the support strand and is paired with the penultimate nucleotide ofthe helper strand; wherein position n is the nucleotide position whichupon ligation of the ligation polynucleotide to the cleaved scaffoldpolynucleotide will be opposite the next nucleotide of the predefinedsequence incorporated in that cycle (step 7), and wherein position n+2is the second nucleotide position in the support strand relative toposition n in the direction proximal to the helper strand/distal to theprimer strand portion.
 14. A method according to claim 13, wherein: (i)in the cleavage step of the first cycle (step 2) the universalnucleotide instead occupies position n+2 in the support strand of thescaffold polynucleotide, wherein n+2 is the second nucleotide positionin the support strand relative to position n in the direction proximalto the helper strand/distal to the primer strand portion; and thesupport strand of the scaffold polynucleotide is cleaved betweenpositions n and n−1; (ii) in the ligation step of the first cycle (step5) the complementary ligation end of the ligation polynucleotide isstructured such that the universal nucleotide instead occupies positionn+3 in the support strand and is paired with the nucleotide which is 2positions removed from the terminal nucleotide of the helper strand atthe complementary ligation end; wherein position n+3 is position 3 inthe support strand relative to position n in the direction proximal tothe helper strand/distal to the primer strand portion; (iii) in thecleavage step of the second cycle (step 6) and in cleavage steps of allsubsequent cycles the universal nucleotide occupies position n+2 in thesupport strand of the scaffold polynucleotide, and the support strand ofthe scaffold polynucleotide is cleaved between positions n and n−1; and(iv) in the ligation step of the second cycle (step 9) and in ligationsteps of all subsequent cycles the complementary ligation end of theligation polynucleotide is structured such that the universal nucleotideoccupies position n+3 in the support strand and is paired with thenucleotide which is 2 positions removed from the terminal nucleotide ofthe helper strand at the complementary ligation end.
 15. A methodaccording to claim 13, wherein: (i) in the cleavage step of the firstcycle (step 2) the universal nucleotide instead occupies position n+2+xin the support strand of the scaffold polynucleotide, wherein n+2 is thesecond nucleotide position in the support strand relative to position nin the direction proximal to the helper strand/distal to the primerstrand portion; and the support strand of the scaffold polynucleotide iscleaved between positions n and n−1; (ii) in the ligation step of thefirst cycle (step 5) the complementary ligation end of the ligationpolynucleotide is structured such that the universal nucleotide insteadoccupies position n+3+x in the support strand and is paired with thenucleotide which is 2+x positions removed from the terminal nucleotideof the helper strand at the complementary ligation end; wherein positionn+3 is position 3 in the support strand relative to position n in thedirection proximal to the helper strand/distal to the primer strandportion; (iii) in the cleavage step of the second cycle (step 6) and incleavage steps of all subsequent cycles the universal nucleotideoccupies position n+2+x in the support strand of the scaffoldpolynucleotide and the support strand of the scaffold polynucleotide iscleaved between positions n and n−1; (iv) in the ligation step of thesecond cycle (step 9) and in ligation steps of all subsequent cycles thecomplementary ligation end of the ligation polynucleotide is structuredsuch that the universal nucleotide occupies position n+3+x in thesupport strand and is paired with the nucleotide which is 2+x positionsremoved from the terminal nucleotide of the helper strand at thecomplementary ligation end; and (v) wherein x is a whole number between1 to 10 or more, and wherein x is the same whole number in steps (2),(5), (6) and (9).
 16. A method according to any one of the precedingclaims, wherein a partner nucleotide which pairs with a first/nextnucleotide of the predefined sequence is a nucleotide which iscomplementary with the first/next nucleotide, preferably naturallycomplementary.
 17. A method according to any one of claims 6 to 16,wherein in any one or more cycles of synthesis, or in all cycles ofsynthesis, prior to step (2) and/or (6) the scaffold polynucleotide isprovided comprising a synthesis strand and a support strand hybridizedthereto, and wherein the synthesis strand is provided without a helperstrand.
 18. A method according to any one of claims 6 to 17, wherein inany one or more cycles of synthesis, or in all cycles of synthesis,prior to step (2) and/or (6) the synthesis strand is removed from thescaffold polynucleotide.
 19. A method according to claim 18, wherein thehelper strand portion of the synthesis strand is removed from thescaffold polynucleotide by: (i) heating the scaffold polynucleotide to atemperature of about 80° C. to about 95° C. and separating the helperstrand portion from the scaffold polynucleotide, (ii) treating thescaffold polynucleotide with urea solution, such as 8M urea andseparating the helper strand portion from the scaffold polynucleotide,(iii) treating the scaffold polynucleotide with formamide or formamidesolution, such as 100% formamide and separating the helper strandportion from the scaffold polynucleotide, or (iv) contacting thescaffold polynucleotide with a single-stranded polynucleotide moleculewhich comprises a region of nucleotide sequence which is complementarywith the sequence of the helper strand portion, thereby competitivelyinhibiting the hybridisation of the helper strand portion to thescaffold polynucleotide.
 20. A method according to any one of claims 4to 7 and claims 16 to 19, wherein each cleavage step comprises a twostep cleavage process wherein each cleavage step comprises a first stepcomprising removing the universal nucleotide thus forming an abasicsite, and a second step comprising cleaving the support strand at theabasic site.
 21. A method according to claim 20, wherein the first stepis performed with a nucleotide-excising enzyme.
 22. A method accordingto claim 21, wherein the nucleotide-excising enzyme is a 3-methyladenineDNA glycosylase enzyme.
 23. A method according to claim 22, wherein thenucleotide-excising enzyme is: i. human alkyladenine DNA glycosylase(hAAG); or ii. uracil DNA glycosylase (UDG).
 24. A method according toany one of claims 20 to 23, wherein the second step is performed with achemical which is a base.
 25. A method according to claim 24, whereinthe base is NaOH.
 26. A method according to any one of claims 20 to 23,wherein the second step is performed with an organic chemical havingabasic site cleavage activity.
 27. A method according to claim 26,wherein the organic chemical is N,N′-dimethylethylenediamine.
 28. Amethod according to any one of claims 20 to 23, wherein the second stepis performed with an enzyme having abasic site lyase activity,optionally wherein the enzyme having abasic site lyase activity is. (i)AP Endonuclease 1; (ii) Endonuclease III (Nth); or (iii) EndonucleaseVIII.
 29. A method according to any one of claims 4 to 7 and claims 16to 19, wherein each cleavage step comprises a one step cleavage processcomprising removing the universal nucleotide with a cleavage enzymewherein the enzyme is (i) Endonuclease III; (ii) Endonuclease VIII;(iii) formamidopirimidine DNA glycosylase (Fpg); or (iv) 8-oxoguanineDNA glycosylase (hOGG1).
 30. A method according to any one of claims 4to 6, claim 8 and claim 9, wherein the cleavage step comprises cleavingthe support strand with an enzyme.
 31. A method according to claim 30,wherein the enzyme cleaves the support strand after the nucleotide whichis next to the universal nucleotide, thereby creating the overhangingend in the synthesis strand.
 32. A method according to claim 30 or claim31, wherein the enzyme is Endonuclease V.
 33. A method according to anyone of claims 4 to 6, claim 8 and claim 9, wherein the cleavage stepcomprises cleaving the support strand with an enzyme.
 34. A methodaccording to claim 33, wherein the enzyme cleaves the support strandafter the nucleotide which is next to the universal nucleotide, therebycreating the overhanging end in the synthesis strand.
 35. A methodaccording to claim 33 or claim 34, wherein the enzyme is Endonuclease V.36. A method according to any one of the preceding claims, wherein bothstrands of the synthesised double-stranded polynucleotide are DNAstrands.
 37. A method according to any one of claims 5 to 36, whereinthe synthesis strand and the support strand are DNA strands.
 38. Amethod according to claim 36 or claim 37, wherein incorporatednucleotides are dNTPs.
 39. A method according to claim 38 whereinincorporated nucleotides are dNTPs comprising a reversible terminatorgroup.
 40. A method according to claim 39, wherein one or more of theincorporated nucleotides comprising a reversible terminator group are3′-O-allyl-dNTPs.
 41. A method according to claim 39, wherein one ormore of the incorporated nucleotides comprising a reversible terminatorgroup are 3′-O-azidomethyl-dNTPs.
 42. A method according to any one ofclaims 1 to 35, wherein a first strand of the synthesiseddouble-stranded polynucleotide is a DNA strand and the second strand ofthe synthesised double-stranded polynucleotide is an RNA strand.
 43. Amethod according to any one of claims 1 to 35 and claim 42, wherein thesynthesis strand is an RNA strand and the support strand is a DNAstrand.
 44. A method according to claim 43, wherein incorporatednucleotides are NTPs.
 45. A method according to claim 44, whereinincorporated nucleotides are NTPs comprising a reversible terminatorgroup.
 46. A method according to claim 45, wherein incorporatednucleotides comprising a reversible terminator group are3′-O-allyl-NTPs.
 47. A method according to claim 45, whereinincorporated nucleotides comprising a reversible terminator group are3′-O-azidomethyl-NTPs.
 48. A method according to any one of claims 1 to41, wherein the enzyme is a polymerase which is a DNA polymerase,preferably a modified DNA polymerase having an enhanced ability toincorporate a dNTP comprising a reversible terminator group compared toan unmodified polymerase.
 49. A method according to claim 48, whereinthe polymerase is a variant of the native DNA polymerase fromThermococcus species 9° N, preferably species 9° N-7.
 50. A methodaccording to any one of claims 1 to 32 and 42 to 47, wherein the enzymeis a polymerase which is an RNA polymerase such as T3 or T7 RNApolymerase, optionally a modified RNA polymerase having an enhancedability to incorporate an NTP comprising a reversible terminator groupcompared to an unmodified polymerase.
 51. A method according to any oneof claims 1 to 47, wherein the enzyme has a terminal transferaseactivity, optionally wherein the enzyme is a terminal nucleotidyltransferase, a terminal deoxynucleotidyl transferase, terminaldeoxynucleotidyl transferase (TdT), pol lambda, pol micro or 129 DNApolymerase.
 52. A method according to any one of claims 4 to 50, whereinthe step of removing the reversible terminator group from the first/nextnucleotide is performed with tris(carboxyethyl)phosphine (TCEP).
 53. Amethod according to any one of claims 4 to 51, wherein the step ofligating a double-stranded ligation polynucleotide to the cleavedscaffold polynucleotide is performed with a ligase enzyme.
 54. A methodaccording to claim 53, wherein the ligase enzyme is a T3 DNA ligase or aT4 DNA ligase.
 55. A method according to any one of claims 6 to 54,wherein in steps (1), (5) and/or (9) the helper strand and the portionof the support strand hybridized thereto are connected by a hairpinloop.
 56. A method according to any one of claims 4 to 54, wherein instep (1) the synthesis strand comprising the primer strand portion andthe portion of the support strand hybridized thereto are connected by ahairpin loop.
 57. A method according to any one of claims 6 to 54,wherein in steps (1), (5) and/or (9): a) the helper strand and theportion of the support strand hybridized thereto are connected by ahairpin loop; and b) the synthesis strand comprising the primer strandportion and the portion of the support strand hybridized thereto areconnected by a hairpin loop.
 58. A method according to any one of claims6 to 57, wherein at least one of the ligation polynucleotides isprovided as a single molecule comprising a hairpin loop connecting thesupport strand and the helper strand at the end opposite thecomplementary ligation end.
 59. A method according to any one of claims6 to 58, wherein the ligation polynucleotides of each synthesis cycleare provided as single molecules each comprising a hairpin loopconnecting the support strand and the helper strand at the end oppositethe complementary ligation end.
 60. A method according to any one ofclaims 6 to 59, wherein in step (1) the synthesis strand comprising theprimer strand portion and the portion of the support strand hybridizedthereto are tethered to a common surface.
 61. A method according toclaim 60 wherein the primer strand portion and the portion of thesupport strand hybridized thereto each comprise a cleavable linker,wherein the linkers may be cleaved to detach the double-strandedpolynucleotide from the surface following synthesis.
 62. A methodaccording to any one of claims 4 to 59, wherein in step (1) the primerstrand portion of the synthesis strand and the portion of the supportstrand hybridized thereto are connected by a hairpin loop, and whereinthe hairpin loop is tethered to a surface.
 63. A method according toclaim 62 wherein the hairpin loop is tethered to a surface via acleavable linker, wherein the linker may be cleaved to detach thedouble-stranded polynucleotide from the surface following synthesis. 64.A method according to claim 61 or claim 63, wherein the cleavable linkeris a UV cleavable linker.
 65. A method according to any one of claims 60to 64, wherein the surface is a microparticle.
 66. A method according toany one of claims 60 to 64, wherein the surface is a planar surface. 67.A method according to claim 66, wherein the surface comprises a gel. 68.A method according to claim 67, wherein the surface comprises apolyacrylamide surface, such as about 2% polyacrylamide, preferablywherein the polyacrylamide surface is coupled to a solid support such asglass.
 69. A method according to any one of claims 60 to 68, wherein thesynthesis strand comprising the primer strand portion and the portion ofthe support strand hybridized thereto are tethered to the common surfacevia one or more covalent bonds.
 70. A method according to claim 69,wherein the one or more covalent bonds is formed between a functionalgroup on the common surface and a functional group on the scaffoldmolecule, wherein the functional group on the scaffold molecule is anamine group, a thiol group, a thiophosphate group or a thioamide group.71. A method according to claim 70, wherein the functional group on thecommon surface is a bromoacetyl group, optionally wherein thebromoacetyl group is provided on a polyacrylamide surface derived usingN-(5-bromoacetamidylpentyl) acrylamide (BRAPA).
 72. A method accordingto any one of claims 4 to 71, wherein the step of removing thereversible terminator group from a nucleotide of the predefined sequenceis performed before or after the ligation step.
 73. A method accordingto any one of the preceding claims, wherein synthesis cycles areperformed in droplets within a microfluidic system.
 74. A methodaccording to claim 73, wherein the microfluidic system is anelectrowetting system.
 75. A method according to claim 74, wherein themicrofluidic system is an electrowetting-on-dielectric system (EWOD).76. A method according to any one of the preceding claims, whereinfollowing synthesis the strands of the double-stranded polynucleotidesare separated to provide a single-stranded polynucleotide having apredefined sequence.
 77. A method according to any one of the precedingclaims, wherein following synthesis the double-stranded polynucleotideor a region thereof is amplified, preferably by PCR.
 78. A method ofassembling a polynucleotide having a predefined sequence, the methodcomprising performing the method of any one of the preceding claims tosynthesise a first polynucleotide having a predefined sequence and oneor more additional polynucleotides having a predefined sequence andjoining together the first and one or more additional polynucleotides.79. A method according to claim 78 wherein the first polynucleotide andthe one or more additional polynucleotides are double-stranded.
 80. Amethod according to claim 78 wherein the first polynucleotide and theone or more additional polynucleotides are single-stranded.
 81. A methodaccording to any one of claims 78 to 80, wherein the firstpolynucleotide and the one or more additional polynucleotides arecleaved to create compatible termini and joined together, preferably byligation.
 82. A method according to claim 81, wherein the firstpolynucleotide and the one or more additional polynucleotides arecleaved by a restriction enzyme at a cleavage site.
 83. A methodaccording to any one of claims 74 to 82, wherein the synthesis and/orassembly steps are performed in droplets within a microfluidic system.84. A method according to claim 83 wherein the assembly steps compriseproviding a first droplet comprising a first synthesised polynucleotidehaving a predefined sequence and a second droplet comprising anadditional one or more synthesised polynucleotides having a predefinedsequence, wherein the droplets are brought in contact with each otherand wherein the synthesised polynucleotides are joined together therebyassembling a polynucleotide comprising the first and additional one ormore polynucleotides.
 85. A method according to claim 84 wherein thesynthesis steps are performed by providing a plurality of droplets eachdroplet comprising reaction reagents corresponding to a step of thesynthesis cycle, and sequentially delivering the droplets to thescaffold polynucleotide in accordance with the steps of the synthesiscycles.
 86. A method according to claim 85, wherein following deliveryof a droplet and prior to the delivery of a next droplet, a washing stepis carried out to remove excess reaction reagents.
 87. A methodaccording to claims 85 and 86, wherein the microfluidic system is anelectrowetting system.
 88. A method according to claim 87, wherein themicrofluidic system is an electrowetting-on-dielectric system (EWOD).89. A method according to any one of claims 85 to 88, wherein synthesisand assembly steps are performed within the same system.
 90. Apolynucleotide synthesis system for carrying out the method according toany one of claims 1 to 89, comprising: (a) an array of reaction areas,wherein each reaction area comprises at least one scaffoldpolynucleotide; and (b) means for the delivery of the reaction reagentsto the reaction areas; and optionally, (c) means to cleave thesynthesised double-stranded polynucleotide from the scaffoldpolynucleotide.
 91. A system according to claim 90 further comprisingmeans for providing the reaction reagents in droplets and means fordelivering the droplets to the scaffold polynucleotide in accordancewith the synthesis cycles.
 92. A kit for use with the system of claim 90or 91 and for carrying out the method according to any one of claims 1to 89, the kit comprising volumes of reaction reagents corresponding tothe steps of the synthesis cycles.
 93. A method of making apolynucleotide microarray, wherein the microarray comprises a pluralityof reaction areas, each area comprising one or more polynucleotideshaving a predefined sequence, the method comprising: a) providing asurface comprising a plurality of reaction areas, each area comprisingone or more double-stranded anchor or scaffold polynucleotides, and b)performing cycles of synthesis according to the method of any one ofclaims 1 to 75 at each reaction area, thereby synthesising at each areaone or more double-stranded polynucleotides having a predefinedsequence.
 94. A method according to claim 93, wherein followingsynthesis the strands of the double-stranded polynucleotides areseparated to provide a microarray, wherein each area comprises one ormore single-stranded polynucleotides having a predefined sequence.